Biosynthesis and Use of Cobalamin (B<sub>12</sub>)
JORGE C. ESCALANTE-SEMERENA1* AND MARTIN J. WARREN2*
[SECTION EDITOR: T. BEGLEY]
Posted August 5, 2008
Department of Bacteriology, University of Wisconsin—Madison, 1550 Linden Drive, Madison, WI 53706,1 and Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom2
*Corresponding authors. Mailing address for Jorge C. Escalante-Semerena: Department of Bacteriology, University of Wisconsin—Madison, 1550 Linden Drive, Madison, WI 53706. Phone: (608) 262-7379, Fax: (608) 265-7909, E-mail:
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. Mailing address for Martin J. Warren: Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom. Phone: 44-1227-824690, Fax: 44-122-776-3912, E-mail:
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.
This review attempts to summarize research performed over the last 23 years on the genetics, enzyme structures and functions, and regulation of the expression of the genes encoding functions involved in adenosylcobalamin (AdoCbl, or coenzyme B12) biosynthesis. We also discuss the role of coenzyme B12 in the physiology of Salmonella enterica serovar Typhimurium LT2 and Escherichia coli.
John Roth and coworkers (first at the University of Utah, Salt Lake City, and now at the University of California, Davis) discovered AdoCbl biosynthesis in serovar Typhimurium in the early 1980s (122). This discovery had a broad and profound impact on the field of B12 biosynthesis research at large. Roth’s elegant use of classical genetic approaches defined the cobalamin biosynthetic (cob) genes on the basis of the phenotypes of mutant strains. The knowledge and use of Cob phenotypes not only were pivotal in the analysis of the cobalamin biosynthetic pathway in enteric bacteria, but also led researchers to the discovery of two superoperons dedicated to the AdoCbl-dependent breakdown of ethanolamine and 1,2-propanediol (1,2-Pdl) in serovar Typhimurium (121, 223, 224), new enzymes and pathways for corrinoid salvaging in archaea (277, 305, 306, 308), the 2-methylcitric acid cycle of propionate catabolism in bacteria (107, 108, 109, 275), and an area of microbial cell physiology previously studied only in the Eucarya domain of life (287).
Roth and coworkers later determined the nucleotide sequence of the B12 biosynthetic genes (226), establishing the organization of 20 genes, providing gene nomenclature that reflected the branch of the pathway in which specific functions were involved, and opening the gates to gene cloning and overexpression, thus facilitating the isolation of proteins in amounts that allowed detailed biochemical and structural studies (55, 194, 199, 209, 239, 280, 304). The discovery of B12 biosynthesis in serovar Typhimurium complemented the extensive and elegant biochemical analyses of the de novo corrin biosynthetic pathway in Pseudomonas pertucinogena (formerly P. denitrificans [3a]) by the Rhône-Poulenc Rorer S.A. research group led by F. Blanche in France (reviewed in reference 22) and by the Cambridge group led by A. Battersby in the United Kingdom and the Texas A&M group led by A. I. Scott in the United States (reviewed in references 14, 16, 241, and 242), which resulted in the realization that two separate pathways representing aerobic and anaerobic routes for de novo cobalamin biosynthesis exist (26).
Much has been learned about the assembly of the nucleotide loop of AdoCbl in serovar Typhimurium. We now understand how incomplete corrinoids (e.g., cobyric acid and cobinamide) are assimilated into AdoCbl (277, 278), and a new intermediate of the lower-ligand activation branch of the pathway has been identified (165), the sequence of the reactions has been reassessed (313), and the in vitro reconstitution of the nucleotide loop assembly (NLA) pathway has been accomplished (166).
Knowledge of how serovar Typhimurium synthesizes the lower ligand base of AdoCbl (i.e., 5,6-dimethylbenzimidazole [DMB]) remains limited. Most of what we know about DMB synthesis in serovar Typhimurium and other bacteria was learned from insightful work by B. Keck, P. Renz, and coworkers (Universität Hohenheim, Stuttgart, Germany) (133, 134, 203), from work with other bacteria (46, 95, 192), and from abiotic chemical synthesis (164). The identity of the genes and gene products involved in DMB synthesis in serovar Typhimurium and E. coli is still an open question. On a more general level, the discovery of the B12 pathway in serovar Typhimurium, a tractable and well-characterized organism, led to the development of complex genetic engineering projects to investigate the roles of individual enzymes within the pathway (194, 210) and a greater understanding of the evolution of complex pathways (197, 225). It also highlighted the importance of horizontal gene transfer in microbial physiology and biochemistry.
In short, John Roth’s seminal contributions to the field of coenzyme B12 biosynthesis research brought the power of classical and molecular genetic, biochemical, and structural approaches to bear on the extremely challenging problem of dissecting the steps of what has turned out to be one of the most complex biosynthetic pathways known. The impact of the contributions that John Roth and his coworkers made and continue to make to this exciting field of microbial physiology is respectfully and enthusiastically acknowledged.
Unfortunately, the B12 field suffers from complex nomenclature. The Commission on Biochemical Nomenclature of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular Biology published a set of recommendations for the nomenclature of corrinoids (114), which was later revised (115). For the reader’s benefit, we define below terminology used throughout this chapter.
Corrinoids. Molecules comprising the cobalt-containing cyclic tetrapyrrole known as the corrin ring
Incomplete corrinoids. Corrinoids lacking the lower (Coα) axial ligand base
Four-coordinate corrinoids. Corrinoids that have the four pyrrolic equatorial ligands but lack axial ligands
Cobamide (Cba). A complete corrinoid, that is, a corrinoid containing both lower and upper ligands. Cobamides are also referred to as B12, without the identification of the nature of the upper ligand
Cobalamin (Cbl). Cobamide whose lower ligand base is DMB
Pseudo-B12 (AdeCba). Cobamide whose lower ligand is adenine, not DMB
Factor III. Cobamide with 5-hydroxybenzimidazole as the lower ligand base
Vitamin B12, or cyanocobalamin (CNCbl). Cbl with a cyano group as the upper (Coβ) ligand
Coenzyme B12, or AdoCbl. Cbl with 5'-deoxyadenosine (Ado) as the upper (Coβ) axial ligand
Coβ (upper) axial ligand. 5-Deoxyadenosine
Coα (lower) axial ligand. DMB in aerobically synthesized AdoCbl. The cobamide synthesized under anaerobic conditions contains either adenine or 2-methyladenine as the lower ligand
Precorrin-n. Intermediate of corrin ring biosynthesis between uroporphyrinogen III (uro’gen III) and cobyrinic acid, where n refers to the number of methyl groups that have been added to the macrocyclic template
Factor n. Oxidized form of precorrin-n
α-Ribazole. Nucleotide loop consisting of dimethylbenzimidazole attached to ribose
Cbl (Fig. 1) is a structurally complex cobalt-containing cyclic tetrapyrrolidine/pyrroline (89) with the following unique features: (i) a highly decorated corrin ring with a cobalt atom held by equatorial coordination bonds to the nitrogens of the pyrroles, (ii) an upper (Coβ) ligand covalently bound to the cobalt, (iii) a lower (Coα) ligand interacting with the cobalt via a pH-dependent coordination bond, (iv) a nucleotide loop that tethers the corrin ring to the lower ligand base, (v) an α-N-glycosidic bond, and (vi) a phosphodiester bond involving the 3' hydroxyl group of the ribosyl moiety of the nucleotide. Cbl is one of only three known coenzymes with a phosphodiester bond; the other two coenzymes are methanopterin and coenzyme F420 (61).
The work leading to the identification of the chemical structure of Cbl and its complete organic synthesis, as well as spectroscopy and crystallographic analyses, fall beyond the scope of this review. Readers interested in these subjects are referred to comprehensive reviews and papers (15, 33, 38, 39, 91, 92, 103, 139, 168, 245, 261, 262, 263, 309).
Cbl is an essential nutrient for animals. In humans, a Cbl-deficient diet leads to severe health problems. For example, impaired absorption of Cbl causes megaloblastic anemia (pernicious anemia), while a diet deficient in Cbl leads to severe nervous system disorders (257). Animals do not synthesize Cbl, but they do convert hydroxocobalamin (OH-Cbl) to AdoCbl (coenzyme B12), the coenzymatic form of Cbl (148). The inability to make this conversion results in methylmalonic aciduria, an inborn disorder of metabolism that gives rise to severe mental retardation and high infant mortality (9). Although there is no evidence that higher plants either use or synthesize cobalamin, many microalgae are cobalamin auxotrophs (56, 57).
To date, Cbl biosynthesis has been found only in prokaryotes, including aerobes, anaerobes, photosynthetic bacteria, fermentative bacteria, and facultative anaerobes of the bacterial and archaeal domains (12, 116, 137, 140, 141, 171, 270, 303, 308). As discussed below, important differences exist in the strategies used by Cbl producers to salvage incomplete corrinoids from their environments.
Serovar Typhimurium and E. coli synthesize AdoCbl from cobinamide (Cbi), an incomplete corrinoid that lacks the nucleotide loop. However, unlike E. coli, serovar Typhimurium can synthesize the corrin ring de novo, albeit only under anoxic conditions (122). The evolution of this metabolic difference has been investigated previously (144, 145, 226). A correlation between AdoCbl biosynthesis and 1,2-Pdl degradation in serovar Typhimurium exists (144, 225), and the results from studies of the coregulation of Cbl biosynthesis and 1,2-Pdl utilization (pdu) genes provide support for this conclusion (30, 218). As mentioned above, serovar Typhimurium and E. coli convert Cbi to AdoCbl. That is, both organisms can attach the 5'-deoxyadenosyl upper ligand to Cbi, synthesize the lower ligand base, and assemble the nucleotide loop (73, 160).
As discussed below, the nature of the lower ligand in serovar Typhimurium varies as a function of oxygen availability in the environment (133, 134).
Cbi salvaging in serovar Typhimurium and E. coli occurs in the presence or absence of oxygen (73, 122), suggesting the involvement of oxygen-labile intermediates and/or enzymes in the de novo corrin ring biosynthetic branch of the pathway. The identity of any oxygen-labile Cob enzyme or intermediate has not been reported.
AdoCbl Synthesis in E.coli.
E. coli synthesizes cobamides if provided with the precursor Cbi (80). At present, E. coli is not considered to be a de novo AdoCbl producer.
Four Cbl-dependent enzymes in serovar Typhimurium have been described. Although none of the functions of these enzymes are essential for cell survival, they have been instrumental in the assignment of Cob protein functions.
MetH.
The methylation of homocysteine is the last step in the synthesis of methionine (96). This reaction is catalyzed by methyltetrahydrofolate-dependent methyltransferases (methionine synthases) that do or do not require Cbl as a cofactor (65). The Cbl-dependent methionine synthase of E. coli (MetH; EC 2.1.1.3) has been studied in great detail (170). In the homocysteine methylation reaction, 5-methyltetrahydrofolate donates a methyl group to cob(I)alamin to generate methyl-Cbl, which in turn donates its methyl group to homocysteine. The crystal structure of the Cbl-binding domain of the E. coli MetH protein was resolved previously (64), and knowledge gained from its structure guided elegant mechanistic studies describing the catalytic events and the reactivation of Cbl to form methyl-Cbl (4, 5, 6, 7, 10, 11, 62, 63, 74, 97, 104, 105, 106, 120, 157, 162, 249).
The methylation of homocysteine by the Cbl-independent enzyme (MetE; EC 2.1.1.14) has also been extensively studied (93, 104, 188, 189, 273, 274, 314), but it is not further discussed here. Genetic analyses of the Cbl biosynthetic pathway are performed with strains carrying a null allele of metE, demanding the use of the Cbl-dependent MetH enzyme in the final step of methionine synthesis (122) and hence providing a tool for the assessment of Cbl synthesis. The Cbl requirement for MetH is met by as little as 10 to 11 M Cbl in the medium (13), making a metE strain an extremely sensitive bioassay indicator strain for corrinoids in the environment. Routine work uses 15 nM corrinoids in the medium.
Ethanolamine Ammonia-Lyase.
Ethanolamine can serve as the sole carbon, energy, and nitrogen source for serovar Typhimurium and E. coli (47, 223). The enzyme ethanolamine ammonia-lyase (also known as ethanolamine deaminase; EC 4.3.1.7) requires AdoCbl as the coenzyme to convert ethanolamine to acetaldehyde and ammonia (8). Ethanolamine ammonia-lyase is encoded by the eutBC genes of the 16-gene ethanolamine utilization (eut) operon (20, 21, 75, 138, 223, 224, 266).
The growth of serovar Typhimurium on ethanolamine as the carbon, nitrogen, and energy source is an additional means to assess AdoCbl synthesis in vivo (223). However, efficient growth on ethanolamine requires 150 nM corrinoids in the medium. This higher-level demand for corrinoids has been used as a stringent assay of the functionality of heterologous genes introduced into serovar Typhimurium strains lacking a specific cob function (43). Note that when incomplete corrinoids such as Cby and Cbi are used instead of Cbl, DMB must be added to the medium; otherwise, growth is poor. The reason for the DMB requirement is likely the subsaturating level of adenosylated Cbi (AdoCbi)-GDP for the AdoCbl-5'-P synthase (CobS) enzyme. CobS product formation is accelerated by increasing the level of endogenous α-ribazole-P, the cosubstrate of the CobS enzyme (313).
1,2-Pdl Dehydratase.
1,2-Pdl also serves as a carbon and energy source for serovar Typhimurium under oxic and anoxic conditions (193). Pdl dehydratase (also known as propane-1,2-diol hydrolyase; EC 4.2.1.28) is the AdoCbl-dependent enzyme that catalyzes the dehydration of 1,2-Pdl into propionaldehyde (282, 284). In serovar Typhimurium, the subunits of Pdl dehydratase are encoded by the pduCDE genes (32), which are part of the 21-gene Pdl utilization (pdu) operon located in the 5' direction from the Cbl biosynthetic operon (31). The expression of the pdu operon is activated by the AraC-type PocR protein in response to 1,2-Pdl in the medium (30, 121, 218). Like growth on ethanolamine, 1,2-Pdl utilization requires >150 nM corrinoids in the medium, providing a third means of assessing the functionality of heterologous B12 biosynthetic genes. The pdu operon is absent in E. coli.
Unlike that of serovar Typhimurium, the E. coli genome contains a putative four-gene operon (yli-argK-ygfG-ygfH) that encodes functions needed to convert succinate to propionate (98, 227). The yliK gene encodes a methylmalonyl coenzyme A (methylmalonyl-CoA) mutase enzyme (EC 5.4.99.2) that interconverts methylmalonyl-CoA and succinyl-CoA, ygfG encodes a methylmalonyl-CoA decarboxylase, and ygfH encodes a propionyl-CoA:succinate CoA transferase. The absence of a propionyl-CoA carboxylase and a methylmalonyl-CoA epimerase led to the conclusion that E. coli may use the enzymes described above to decarboxylate succinate. At the time of this writing, the metabolic context of this cycle was unclear.
Most of the Cbl biosynthetic genes of serovar Typhimurium are clustered in a 20-gene operon spanning 15,119 bp in the region from bp 2097303 to 2112422 of the genome (226); the promoter region is located between the cbiA and pocR genes (1,977 bp) (Fig. 2).
The first 17 genes of the operon are known as the cbi genes (cbiABCDETFGHJKLMNQOP), for AdoCbi-P synthesis. The cbi functions are necessary but not sufficient for the synthesis of the corrin ring (40). The last three genes of the operon are the cobUST genes, which encode enzymes that catalyze the late steps of AdoCbl biosynthesis. This branch of the pathway is also known as the NLA pathway (166). cobUST functions are necessary but not sufficient for the assembly of the nucleotide loop. One additional gene (cobC), located outside of the operon, is needed to convert AdoCbl-P to AdoCbl, the final product of the pathway (185).
Four additional functions encoded by genes outside of the operon (namely, fldA, fpr, cobA, and cobD) are needed to synthesize AdoCbi-P (40, 73, 78, 94, 271). (Fig. 2). The only Cbl biosynthetic genes of serovar Typhimurium that have not been identified are the one encoding the DMB synthase enzyme and the one encoding l-Thr kinase. A functional homologue of the DMB synthase in other bacteria is BluB, a recently identified oxygenase that converts reduced flavin mononucleotide (FMNH2) to DMB in Rhodospirillum rubrum (95). Although serovar Typhimurium and E. coli have bluB orthologues in their genomes (i.e., nfnB, ydjA, and snrA), evidence that these genes encode DMB synthase has not been reported.
The E. coli K-12 genome does not contain any cbi genes, but it does carry cobUST (bp 2061412 to 2063788) and cobC (bp 668519 to 669130). Note that the cobC gene in the E. coli CFT073 genome is annotated as phpB (for phosphohistidine protein B). All E. coli strains sequenced thus far lack the cobDl-threonine O-3-phosphate decarboxylase (l-threonine O-3-phosphate carboxy-lyase; EC 4.1.1.81) (40), consistent with the absence of the cbiB gene encoding the AdoCbi-P synthase enzyme [adenosylcobyric acid:(R)-1-aminopropan-2-oyl phosphate ligase (ADP forming); EC 6.3.1.10] that uses the product of the CobD reaction [i.e., (R)-1-aminopropan-2-oyl phosphate] as the substrate in the last step of the de novo corrin ring biosynthesis branch of the pathway.
Biosynthesis of uro’gen III.
The corrin ring component of vitamin B12 is a modified tetrapyrrole and belongs to the same family of compounds as heme and siroheme, which are also made by serovar Typhimurium and E. coli. However, vitamin B12 differs from the latter two compounds in three important ways: firstly, the corrin ring of B12 has undergone contraction, losing one of its bridging carbon atoms that join rings A and D (Fig. 1). More detail on the molecular processes that give rise to this ring contraction is given below. Secondly, the metal ion at the center of the core of the macrocycle is cobalt rather than iron. Cobalt is an odd choice of metal ion since it is one of the least abundant life metals on earth and is found in only sparingly soluble quantities in aquatic environments (83). The uptake of cobalt therefore represents a major recruitment challenge to the cobalamin-biosynthesizing bacteria. Finally, the last difference between these modified tetrapyrroles is the structural complexity of B12, in which the corrin ring is decorated by extensive methylation and the side chains are amidated. Unlike all other modified tetrapyrroles, vitamin B12 uniquely provides both upper and lower axial ligands for the centrally chelated metal ion. For this part of the story, the construction of the corrin ring component of cobalamin will be described, with the initial emphasis on the biogenesis of the tetrapyrrole framework (Fig. 3). All modified tetrapyrroles are based upon the unsymmetrical uro’gen III template, so the synthesis of this molecular scaffold will be described first.
In E. coli and serovar Typhimurium, uro’gen III represents the first branch point in the pathway, where the routes for cobalamin and siroheme synthesis diverge from that for heme synthesis. The biosynthesis of uro’gen III requires six enzyme-mediated reactions, starting from glutamic acid (Fig. 3). The identification of glutamate as a tetrapyrrole precursor in plants was first reported in 1974 (19), and the same arrangement was shown later to occur in E. coli (153). In the first of these reactions, which is common to the synthesis of proteins, glutamate is charged with its tRNA to generate the aminoacyl-glutamyl-tRNA derivative (Fig. 3) (118, 119, 132, 288). The HemA protein acts on this substrate and reduces it to glutamate semialdehyde (GSA) in a reaction that requires NADPH (Fig. 3). Both the E. coli and serovar Typhimurium enzymes have been difficult to study due to their susceptibility to proteolytic breakdown, which is a reflection of the regulation of HemA mainly by conditional protein stability (293, 294, 295). The structure of HemA from the archaeon Methanopyrus kandleri has been resolved, revealing an elongated V-shaped homodimer with distinct modular domains (176). An investigation into the mechanism of the E. coli HemA has shown that it involves a process that advances via a thioester intermediate utilizing a reactive cysteine residue at the active site of the enzyme (234).
An aminomutase reaction results in the conversion of GSA into 5-aminolevulinic acid (ALA) (Fig. 3) in a reaction catalyzed by GSA aminotransferase (GSA-AT), which is encoded by hemL (67, 111, 112). As would be expected for an aminotransfer reaction, this enzyme employs pyridoxal phosphate as a prosthetic group and the reaction starts and finishes with the cofactor in its pyridoxamine form. Investigations of the E. coli HemL have shown that the reaction is likely to proceed from GSA via 4,5-diaminovalerate to ALA (111). The structure of GSA-AT from Synechococcus sp. revealed that the protein belongs to the aspartate aminotransferase family (102). Moreover, it has been proposed that GSA-AT may form a complex with the HemA protein in which the homodimer GSA-AT fits into the large groove of the V-shaped HemA, presumably to facilitate direct metabolite transfer (channeling) of the potentially reactive GSA (158). It is also interesting that there may be an alternative pathway to ALA in serovar Typhimurium. It has been noted that siroheme and vitamin B12 are both made in small quantities under anaerobic conditions by hemA and hemL mutants, indicating that this second pathway would be independent of the HemA and HemL functions (68).
ALA is dimerized by the action of the ALA dehydratase (ALAD), an enzyme that is encoded by hemB (154). This homo-octameric enzyme utilizes a catalytic zinc ion as a Lewis acid to help promote the Knorr-type condensation between two molecules of ALA to generate the pyrrole porphobilinogen (PBG) (Fig. 3) (173, 254, 255). In ALADs from other organisms, there is no requirement for zinc, but magnesium would appear to play a role in stimulating activity (173, 254, 255). In the E. coli ALAD, magnesium also stimulates activity, but only if zinc is already present (173, 254, 255). The crystal structure of HemB reveals that ALAD is formed from pairs of dimers, in which the Mg is located at the interface of the dimers (70). Single-turnover experiments have shown that the first substrate molecule to bind to the enzyme ultimately forms the propionate half of the product PBG and that the second substrate molecule forms the acetate half of PBG (127). The active site contains two invariable lysine side chains, and structural evidence suggests that catalysis involves a double Schiff base mechanism (69).
Four molecules of PBG are next polymerized by the enzyme PBG deaminase, encoded by hemC, to generate a linear tetrapyrrole called hydroxymethylbilane (Fig. 3). This monomeric enzyme utilizes a novel dipyrromethane cofactor that is made from two molecules of the substrate PBG (99, 129). The cofactor acts as a primer to initiate the polymerization process. The first substrate binds at the active site and is deaminated to generate an azafulvene that then links to the free alpha position of the cofactor. This process is repeated three more times to generate a linear tetrapyrrole joined to the dipyrromethane cofactor (298). The link between the first substrate unit and the tetrapyrrole is hydrolyzed to yield the product and free enzyme. Thus, the enzyme adds the four pyrrole units in an ordered and sequential fashion (128). The crystal structure of the E. coli HemC, which was the first tetrapyrrole biosynthetic enzyme to be identified, reveals that the polypeptide chain is folded into three domains that surround the active site of the enzyme (156).
Uro’gen III is synthesized from hydroxymethylbilane by the enzyme uro’gen III synthase (Fig. 3), which is encoded by hemD (17). This enzyme not only cyclizes the bilane but also inverts the terminal D ring to generate the type III isomer. It seems likely from results of inhibitor studies that the inversion of this D ring involves the formation of a spiro intermediate (260). The crystal structure of the human uro’gen III synthase has been resolved to reveal a protein containing two domains connected by a beta-ladder (169). The active site appears to be located between the domains, but it is still not clear how the enzyme catalyzes this remarkable inversion and cyclization.
Biosynthesis of Precorrin-2, Sirohydrochlorin, and Siroheme.
Uro’gen III is directed away from heme synthesis by the bismethylation of this first macrocyclic intermediate at positions 2 and 7, which generates a dipyrrocorphin called precorrin-2 (also known as dihydrosirohydrochlorin) (Fig. 4) (24, 301). Most cobalamin biosynthetic operons contain a gene that encodes an S-adenosyl-l-methionine (SAM)-dependent methyltransferase for the synthesis of precorrin-2 (58, 207). However, presumably as some artifact of horizontal transfer, this gene is missing from the serovar Typhimurium repertoire (144). Instead, serovar Typhimurium has to rely on CysG as its source of precorrin-2 (122, 123). CysG is a multifunctional protein that is able to catalyze the transformation of uro’gen III into siroheme (253). The CysG gene is clustered with a number of nir genes associated with NADH-dependent nitrite reductase activity, for which siroheme is required as a prosthetic group (187). The E. coli CysG has been extensively studied and was shown to function by transferring two methyl groups from SAM, firstly to C-2 and subsequently to C-7 of the tetrapyrrole framework (300). In the presence of large quantities of the enzyme, a third methyl group is also transferred to C-12 to generate what is thought to be a nonphysiological trimethylpyrrocorphin (301). The primary structure of CysG was found to be significantly longer than that of the uro’gen III methyltransferase isolated from the aerobic cobalamin producer P. denitrificans (in which it is encoded by cobA), with an extra 200 amino acid residues at the N terminus (58, 187). Gene dissection experiments revealed that the uro’gen methyltransferase activity is housed solely in the C-terminal region of the protein (297). Moreover, the whole CysG was found to be further capable of transforming precorrin-2 into siroheme (253). This transformation is accomplished by the NAD-dependent removal of two protons and two electrons to give sirohydrochlorin and by the insertion of ferrous iron to yield siroheme (Fig. 4). However, as the C-terminal region of the protein is not able to catalyze the latter two functions, it was concluded that the dehydrogenase and chelatase activities must be housed within the first 200 amino acids of the N terminus of the protein (304). This led to the idea that CysG represents a fusion between a uro’gen III methyltransferase and a precorrin-2 dehydogenase/ferrochelatase (297). In some organisms, such as Saccharomyces cerevisiae, CysG exists as two distinct proteins (196), whereas in some bacteria, three individual enzymes catalyzing methylation, dehydrogenation, and ferrochelation have been found to be required for siroheme synthesis (195). The crystal structure of the serovar Typhimurium CysG has been determined, and the protein has been found to function as a homodimer, containing, as expected, two structurally independent modules: a C-terminal bismethyltransferase and an N-terminal dual-function dehydrogenase-chelatase (267). The methyltransferase domain of the protein is similar to the uro’gen III methyltransferase enzyme from P. denitrificans, and the presence of S-adenosyl-l-homocysteine within the crystal structure helps define the activity of this region of the protein. Mutations in some residues lead to the accumulation of a monomethylated intermediate (precorrin-1), although the molecular basis for bismethylation has yet to be fully determined (267, 289). The N terminus of the protein is similar to Met8p, the bifunctional precorrin-2 dehydrogenase and sirohydrochlorin ferrochelatase from Saccharomyces cerevisiae (238). Significantly, the structure shows that CysG is a phosphoprotein, although how the protein is phosphorylated has not been investigated. Results from in vitro studies on the serine that is phosphorylated suggest that phosphorylation may act to modulate metabolic flux between the siroheme and cobalamin pathways (267).
Cobalt Insertion Is the First Committed Step in Corrin Ring Synthesis.
Earlier work on the molecular genetics of vitamin B12 biosynthesis in serovar Typhimurium had shown that cysG was required for the biosynthesis of cobalamin (122, 123). Initially, it was thought that CysG was required solely for the synthesis of precorrin-2 and that cobalt was subsequently inserted into precorrin-2 to give cobalt–precorrin-2. However, evidence that CysG also mediates this insertion of cobalt has been presented (76). The fact that serovar Typhimurium cysG mutants fail to synthesize B12 but still make siroheme lends support to this idea. Nonetheless, it was also revealed that there is a specific cobaltochelatase encoded within the main cobalamin operon (199). This enzyme, CbiK, was shown to be able to insert cobalt both in vivo and in vitro. Moreover, it can also insert ferrous iron into sirohydrochlorin to give siroheme, albeit less effectively than CysG (199). The structure of the serovar Typhimurium CbiK revealed topology very similar to that of the ferrochelatase associated with heme synthesis, despite a lack of any significant overall similarity in protein primary structure (239). CbiK is composed of two domains, and the junction between the two domains forms the active site of the enzyme. Two active-site histidines are thought to be involved in the removal of two protons and also in the binding and insertion of Co2+. The two domains of CbiK are also structurally related, suggesting that the protein may have arisen from a gene duplication and the fusion of a smaller gene encoding a more primitive enzyme. Such a smaller type of chelatase has been reported to exist in the vitamin B12 pathway found in the Archaea (34). A more detailed analysis of the specificity for the cobaltochelatase suggested that the true substrate for the enzyme is sirohydrochlorin and not precorrin-2 (149). Thus, CbiK inserts cobalt into sirohydrochlorin to give cobalt-sirohydrochlorin (also known as cobalt factor II) (Fig. 4). Since sirohydrochlorin is one of the products of the CysG-mediated reactions, CysG clearly has a dual function in vitamin B12 biosynthesis in that it not only methylates uro’gen III but also catalyzes the dehydrogenation to give sirohydrochlorin. This product then acts as the substrate for CbiK, which in the presence of Co2+ generates cobalt-sirohydrochlorin (Fig. 4).
Transformation of Cobalt-Sirohydrochlorin into Cobyric Acid.
The cobalamin biosynthetic pathway in P. denitrificans was the first to be elucidated (60), but it was soon realized that there are at least two routes for cobalamin biosynthesis, representing aerobic and anaerobic variations (Table 1) (299). Although there are many similarities between the two pathways, they are genetically distinct (26, 197). The differences in the pathways relate to the timing of cobalt insertion and the method employed for ring contraction. In the aerobic pathway, cobalt is inserted at a late stage and molecular oxygen is required to mediate the ring shrinkage. Conversely, in the anaerobic route, cobalt is inserted at an early stage and contraction is not oxygen dependent. Nonetheless, the changes to the periphery of the corrin molecule, in terms of methylation, decarboxylation, and amidation, occur in the same temporal and spatial orders. Thus, although there are a number of unique enzymes in each pathway, there are also many enzymes that show high degrees of sequence similarity. Indeed, the presence of these homologues assisted the initial deciphering of the pathway in serovar Typhimurium, as based on sequence comparisons, the enzyme functions could be predicted according to the known functions of enzymes elucidated in the aerobic pathway of P. denitrificans (226). The transformation of cobalt-sirohydrochlorin into cobyric acid requires the action of 10 gene products, and the functions of many of these were initially inferred from sequence comparisons to the enzymes with known functions in the aerobic pathway (299).
Table 1Protein requirements and intermediates in the synthesis of cobalamin in P.denitrificans and serovar Typhimurium |
It was based on such a comparison that the C-20 methyltranferase was identified as CbiL (226). In total, eight methyl groups are added to the corrin scaffold during its de novo biosynthesis, although only seven of these SAM-derived methyl groups are present on B12 since the methyl group added to C-20 is lost during the ring contraction process. Eight methyl groups are added by the action of seven separate methyltransferases (only seven are needed, as the first enzyme, CysG, adds two methyl groups). Many of these methyltransferase enzymes show high degrees of sequence similarity, indicating that they have arisen from a common ancestor, probably via a retrograde process of pathway evolution. CbiL shows sequence homology to CysG, CbiH, CbiF, and CbiE, and results from structural work (see below) suggest that all these enzymes belong to a distinct class III family of methyltransferases (236). Initial studies revealed that the serovar Typhimurium CbiL is able to methylate precorrin-2 to give precorrin-3 but with very low yields (209). Subsequently, the enzyme was shown to have an absolute requirement for a metal within the tetrapyrrole substrate (256). Thus, CbiL has greater activity with cobalt–precorrin-2 and cobalt-sirohydrochlorin but will also methylate a number of other transition metal derivatives, including zinc and nickel complexes. However, the enzyme would appear not to discriminate based on the oxidation state of the metal and methylates both Co(II) and Co(III) forms. Recently, the crystal structure of a putative CbiL from Chlorobium tepidum was reported, although no functional work was undertaken (291). A more detailed analysis of a characterized CbiL from Methanothermobacter thermautotrophicus is under way, and this investigation is helping to provide an understanding of how such enzymes are able to catalyze the regiospecific methylation of the corrin framework (82). The conclusion from these studies is that CbiL methylates cobalt-sirohydrochlorin to give cobalt factor III (Fig. 5).
Recent work has led to much greater insight into the series of events that lead to ring contraction and the synthesis of the corrin ring structure (208). Both cobalt–precorrin-3 and cobalt factor III are able to act as substrates for CbiH (230, 231), the enzyme that catalyzes the methylation of C-17. However, the enzyme also catalyzes the delta-lactonization on ring A, and together, these activities result in the contraction of the macrocycle (Fig. 5). There is clearly some controversy over the oxidation state of the true substrate, but as all the studies have been conducted with reactions in crude cell extracts of recombinant E. coli strains overproducing CbiH, there is always the possibility that nonspecific oxidoreductases allowed the interconversion of cobalt–precorrin-3 and cobalt factor III (230). Similarly, it has not been possible to monitor the oxidation state of the cobalt ion, but in most of these studies, it would appear to have been added in the Co(III) state.
Cobalt–precorrin-4 is converted into cobalt–precorrin-5A in a reaction catalyzed by CbiF, which adds a methyl group to C-11 (Fig. 5) (130). Previous research has shown that CbiF is the C-11 methyltransferase, as the enzyme was found to methylate precorrin-3 out of sequence to yield a novel compound termed 4X (209). This nonphysiological intermediate may be an artifact of a high enzyme concentration and exposure to a substrate that does not normally appear in the anaerobic pathway. CbiF was also the first of the cobalamin biosynthetic methyltransferases to have its structure determined, revealing a kidney-shaped molecule that defined a new class of methyltransferases (class III) (236, 240). In class III methyltransferases, the active site is secluded in a cleft between two αβα domains, each containing five strands and four helices, and is defined in part by the presence of S-adenosyl-l-homocysteine which is bound in a tightly folded conformation. In a previous study, cobalt–precorrin-5A was produced by incubating recombinant cell extracts that contained serovar Typhimurium CbiH and CbiF with cobalt factor III (130). The intermediate contained the delta-lactone on ring A and a methyl group at C-11. Surprisingly, when CbiG was then added to the extract, an intermediate called cobalt–precorrin-5B was generated, in which the lactone had been opened and from which the extruded C-2 unit, derived from the methylated C-20 position, was eliminated as acetaldehyde (Fig. 5). The released C-2 unit had been characterized previously as acetaldehyde (292) and is different from the extruded C-2 unit from the aerobic pathway, which is lost as acetic acid. These experiments revealed the function of CbiG for the first time and, as CbiG displays some similarity to an enzyme of the aerobic pathway, CobE, for which no role had been assigned, led to the suggestion that CobE may fulfill a similar task in the aerobic pathway (208).
Cobalt–precorrin-5B is methylated at C-1 to generate cobalt–precorrin-6A by the action of CbiD (Fig. 5) (208). Although the reaction has not been demonstrated directly, the activity of the enzyme has been deduced from several pieces of evidence. Firstly, the enzyme known to catalyze this reaction in the aerobic pathway (CobF) is missing in the anaerobic route, but in all pathways that are missing CobF, a quite different enzyme, CbiD, is always found (198). Secondly, in a genetic engineering study, it was observed that the overproduction of the serovar Typhimurium CbiA, CbiC, CbiD, CbiE, CbiT, CbiF, CbiG, CbiH, CbiJ, CbiK, CbiL, and CbiP proteins resulted in the accumulation of cobyrinic acid a,c-diamide. However, when cbiD was omitted from the artificial operon, 1-desmethylcobyrinic acid a,c-diamide was produced, indicating that CbiD is involved in C-1 methylation (211). Unlike all the other methyltransferases associated with cobalamin biosynthesis, CbiD does not belong to a recognized class of methyltransferases.
By analogy to the aerobic pathway, cobalt–precorrin-6A is likely to be reduced, in an NADPH-dependent process, to cobalt–precorrin-6B by the action of CbiJ (Fig. 5) (208, 299), the homologue of CobK of the aerobic route, whose function has been described previously (27). The next series of reactions involves methylation at C-5 and C-15, as well as the decarboxylation of the acetate side chain at C-12. In the aerobic pathway, the multifunctional CobL enzyme catalyzes all of these reactions (25). However, in serovar Typhimurium, this protein was found to be encoded by two separate genes, cbiE and cbiT (226). Because CbiE has some similarity to the canonical class III methyltransferases, such as CysG, CbiL, CbiH, and CbiF, it was assumed that CbiE would catalyze the methylation reactions at C-5 and C-15 and that CbiT would catalyze the decarboxylation reaction. However, as part of a structural genomic project, the CbiT protein from Methanothermobacter thermautotrophicus was crystallized, was found to have a structure similar to those of class II methyltransferases, and moreover, was able to bind SAH (135). It became apparent, therefore, that this enzyme might be involved in the methylation of C-5 or C-15 and/or the decarboxylation of the acetate side chain attached to C-12. The role of CbiT was shown experimentally when crude cell extracts containing recombinant serovar Typhimurium CbiH, CbiF, CbiG, and CbiT exhibited the accumulation of corrinoid derivatives with a methyl group at C-15 and others with a methyl group at C-15 and a decarboxylated C-12 side chain (230). These results strongly imply that CbiT is responsible for both the methylation at C-5 and the decarboxylation of the acetate side chain and that, as no unmethylated decarboxylated derivatives were isolated, the order of the reaction is methylation followed by decarboxylation (230). Thus, cobalt–precorrin-6B is likely to be acted upon by CbiE to give cobalt–precorrin-7, which in turn acts as the substrate for CbiT to give cobalt–precorrin-8 (Fig. 5).
The formation of cobyrinic acid is completed by CbiC, which catalyzes the rearrangement of the methyl group, previously transferred by CbiF, from C-11 to C-12 (Fig. 6) (208, 299). Again, the activity of this enzyme has not been shown in isolation, but the degree of sequence similarity to the homologous enzyme from the aerobic pathway, CobH, is high, and the recently determined structure of CbiC (310) is very similar to the structure of CobH (246). Cobyrinic acid is subsequently transformed into cobyric acid by the addition of glutamine-derived amide groups to carboxylic acid side chains a, b, c, d, e, and g. Initially, CbiA amidates the a and c side chains to generate cobyrinic a,c-diamide (Fig. 6). Mechanistic investigations into the serovar Typhimurium CbiA have demonstrated that CbiA catalyzes the sequential amidation of the c- and a-carboxylate groups of cobyrinic acid via the formation of a phosphorylated intermediate (84). The final step in cobyric acid synthesis is catalyzed by CbiP (Fig. 6). This enzyme has also been investigated mechanistically, and a time course for the multiple amidation reactions demonstrates that the partially amidated products are released from the enzyme after every round of catalysis (302). Moreover, an analysis of the partially amidated intermediates revealed that the four carboxylate groups are amidated in a specific sequence in which the carboxylate on side chain e is the first to be amidated, followed in turn by d, b, and g. CbiA and CbiP also seem to play a role in helping to orchestrate the corrin biosynthetic pathway. This deduction is based upon the observation that an artificial operon comprising all the cbi genes encoding the enzymes required for the synthesis of cobyric acid requires the presence of both cbiA and cbiP for the incorporation of the C-1 methyl group, suggesting that these genes form a complex with cbiD (210). It is not clear at which point the corrin ring becomes adenosylated. However, a mass spectral analysis of cobyric acid isolated from a recombinant strain of E. coli containing all the cbi genes required for cobyric acid biosynthesis revealed that the compound was already adenosylated (200). This finding does not identify when the cobalt is adenosylated but highlights that the adenosylation takes place by the time cobyric acid is synthesized. For the purposes of this review, we have suggested that cobyric acid is adenosylated to give adenosylcobyric acid (Fig. 6), but it may be an earlier intermediate that is acted upon by the adenosyltransferase (CobA) (see "Attachment of the Upper Ligand to the Cobalt Atom of the Corrin Ring," below).
Biosynthesis of Cobinamide Phosphate.
Cobinamide biosynthesis involves the attachment of the aminopropanol linker chain, which is derived from threonine, to the only remaining carboxylic acid (f) side chain of cobyric acid (Fig. 6). The enzyme that catalyzes this attachment is likely to be CbiB, since mutations in the corresponding gene give rise to the accumulation of cobyric acid (194). Moreover, in the aerobic pathway, the attachment of (R)-1-amino-2-propanol is reported to be catalyzed by two factors, termed α and β, in the presence of ATP (22). The α component was purified to homogeneity and was found to comprise a 38-kDa protein that was not encoded by any known gene of cobalamin biosynthesis. The β component was also purified but found to be a large multiprotein complex with a mass greater than 1,000 kDa containing both CobD and CobC. CobD has a high degree of similarity to CbiB, and it is this component that is thought to be responsible for cobinamide synthesis (225). The P. denitrificans CobC was found to have some homology to type II aminotransferases and also displays similarity to the serovar Typhimurium CobD, which was subsequently shown to be involved in the synthesis of the aminopropanol component. In fact, the serovar Typhimurium CobD is a novel enzyme with l-threonine O-3-phosphate decarboxylase activity that generates (R)-1-amino-2-propanol O-2-phosphate (Fig. 6) (40). The structure of the serovar Typhimurium CobD has been resolved, revealing the native protein to exist as a dimer in which each subunit consists of a large and a small domain (51). As predicted, it is very similar to members of the family of aspartate aminotransferases. In particular, the active site is most related to that observed in histidinol phosphate aminotransferase, suggesting that CobD and histidinol phosphate aminotransferase may be evolutionarily related. The structures of CobD in its apo state, the apo state of CobD in a complex with the substrate, and the product of CobD, the external aldimine complex, have also been determined, which has allowed a proposal as to how the enzyme is able to direct the breakdown of the external aldimine towards decarboxylation instead of amino transfer (54). Cobalamin biosynthesis in serovar Typhimurium cobD mutants can be restored by the addition of exogenous (R)-aminopropanol, suggesting that a kinase phosphorylates the molecule prior to its incorporation into cobyric acid (40).
The conversion of vitamin B12 (CNCbl) to AdoCbl (its biologically active coenzymatic form) requires the attachment of an Ado group as the Coβ upper ligand (Fig. 1). Our current understanding of the corrinoid adenosylation pathway of serovar Typhimurium (and likely of E. coli) is schematized in Fig. 7. Key to this process is the generation of the Co1+ super nucleophile needed to capture the Ado moiety of ATP (110, 290). In vitamin B12, the Co3+ ion is reduced by two one-electron reductions to Co1+ prior to adenosylation.
Strains defective in cobalt ion reduction have not been reported. The failure to isolate mutations in any given gene is attributed to the following possible factors: (i) the enzymes of interest are involved in more than one cellular process, (ii) redundancy exists, (iii) the enzymes are essential, and (iv) an enzyme is not needed.
In all cases discussed below, the addition of AdoCbl or adenosylated precursors such as adenosylated Cby and AdoCbi corrects the effect of the lack of an adenosyltransferase function.
Three-dimensional crystal structures of enzymes involved in corrinoid adenosylation have been reported and are accessible from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB [http://pdbbeta.rcsb.org/pdb/explore/images.do?structureId=1SZQ]). PDB codes for serovar Typhimurium and E. coli enzymes involved in corrin ring biosynthesis, corrinoid adenosylation, and the late steps of the pathway are listed in Table 2.
Table 2PDB codes for enzymes involved in AdoCbl synthesis in serovar Typhimurium and E.coli |
Reduction of Co(III)rrinoids to Co(II)rrinoids.
Reverse genetic approaches to the isolation of the co(III)rrinoid reductase identified the NAD(P)H:flavin oxidoreductase (Fre; EC 1.6.8.1) as the putative co(III)rrinoid reductase (79). This finding was misleading because cob(III)alamin is spontaneously reduced to cob(II)alamin by FMNH2 in the absence of Fre. The latter finding led to the conclusion that the reducing environment inside the cell most likely converts cob(III)alamin to cob(II)alamin upon entry into the cytosol (79). The comparison of the redox potentials at pH 7 for flavin mononucleotide-FMNH2 (−0.19 V [275]), flavin adenine dinucleotide-reduced flavin adenine dinucleotide (−0.22 V [276]), and cob(III)alamin-cob(II)alamin (−0.04 V [152]) lends support to the idea that chemical, not enzymatic, processes generate cob(II)alamin intracellularly. This argument would explain why researchers have not identified strains lacking co(III)rrinoid reductase activity, i.e., the gene encoding such activity does not exist in serovar Typhimurium or in E. coli.
The Housekeeping Adenosyltransferase.
Genetic approaches to the isolation of serovar Typhimurium strains defective in the corrinoid adenosylation pathway identified the cobA gene, which encodes the housekeeping ATP:co(I)rrinoid adenosyltransferase enzyme (EC 2.5.1.17) that catalyzes the formation of the unique covalent Co-C bond between the upper ligand and the corrin ring (Fig. 1) (73). The cobA homologue in E. coli (btuR) was identified in a search for functions involved in the repression of the expression of the btuB and metE genes, encoding the outer membrane corrinoid transport protein BtuB and the Cbl-independent methionine synthase (MetE) enzyme, respectively (159). Based on the observed regulatory phenotype caused by the repression mutations in that study, the gene affected by the mutations was named btuR. This is an unfortunate misnomer because the effect of the lack of btuR function on btuB and metE gene expression is indirect (see below). In fact, BtuR is the ATP:co(I)rrinoid adenosyltransferase of E. coli (160) and is 89% identical at the amino acid level to the serovar Typhimurium CobA protein (272).
The cobA gene is the promoter-distal open reading frame of a two-gene operon (yciK-cobA) whose expression is constitutive. In serovar Typhimurium, CobA activity is needed for the de novo synthesis of the corrin ring and for the salvaging of incomplete and complete corrinoids (73), reflecting the lax specificity of the enzyme for its corrinoid substrate. At present, the identity of the intermediate of the de novo corrin ring biosynthetic pathway that is a substrate of CobA is unknown. Statements in the literature about cobyrinic a,c-diamide’s being the substrate of CobA are not supported by experimental data.
CobA is the best-studied adenosyltransferase (77, 271). Crystal structures of apo CobA and CobA in complexes with substrates have been reported (18). The topology of CobA is surprisingly similar to that of the RecA, helicase domain, F1-ATPase, and the CobU enzyme of serovar Typhimurium; the significance of this similarity is unclear.
CobA has a P loop (37, 150, 232, 251) to which Mg-ATP binds; however, in CobA the P loop is one residue shorter than the consensus (GNGKGKT, not GX4GKT/S) and Mg-ATP binds to it in the orientation opposite that in which it binds to all other nucleotide hydrolases. This unusual orientation of ATP is needed to transfer the adenosyl moiety rather than the γ-phosphate (18).
Lowering the Thermodynamic Barrier to Co2+-to-Co1+ Reduction.
The reduction of Co2+ to Co1+ is thermodynamically challenging for the cell, as indicated by the very low redox potential of the cob(II)alamin-cob(I)alamin couple (−0.61 V [151]). The cellular reductant for the cob(II)alamin-to-cob(I)alamin reaction is likely to be reduced flavodoxin A (78). The reduction of flavodoxin requires the ferredoxin:NADP+ oxidoreductase enzyme (Frp; EC 1.18.1.2) and NADPH+ + H+ as the reductant. No genetic evidence supporting the role of FldA has been reported. The thermodynamic barrier to the Co(II)-to-Co(I) reduction is lowered upon the binding of the co(II)rrinoid substrate to the adenosyltransferase bound to Mg-ATP (see below).
Detailed computational, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy studies of free corrinoids and corrinoids bound to CobA have shed light onto the mechanism of catalysis (262, 263). Magnetic circular dichroism and electron paramagnetic resonance studies of cob(II)alamin bound to CobA in a complex with ATP have provided insights into how the redox barrier to the Co(II)-to-Co(I) reduction is likely to be lowered (264). Analyses of spectral correlations and density functional theory computations revealed that cob(II)alamin or cob(II)inamide bound to CobA-ATP lacks axial bonding interactions, making it an unprecedented four-coordinate corrinoid. The removal of axial ligands stabilizes the 3dz2 molecular orbital of Co2+ and raises the Co2+-Co1+ reduction potential by ~250 mV. This stabilization allows the transfer of one electron from the FldA semiquinone (−440 mV) to the Co(II) ion (264). How the enzyme generates the four-coordinate co(II)rrinoid remains under investigation.
Biochemical and mutational studies of the interactions between FldA and CobA have identified some residues of CobA needed for docking but not for catalysis (41). Mutational analysis is required to investigate how the enzyme generates the four-coordinate corrinoid species.
At present, only one additional CobA-type enzyme from the methanogenic archaeon Methanosarcina mazei strain Gö1 has been isolated and biochemically characterized (43). The archaeal enzyme compensates for the lack of CobA function during the growth of a serovar Typhimurium cobA strain under conditions that demand the synthesis of AdoCbl (43). Unlike that of the serovar Typhimurium enzyme, the activity of the archaeal CobA is stimulated by Ca(II) ions and is inhibited by Mn2+ ions. Both serovar Typhimurium CobA and Methanosarcina mazei CobA enzymes yield triphosphate as the reaction by-product (43, 77).
The PduO and EutT Adenosyltransferases.
The pdu operon of serovar Typhimurium (not present in E. coli) encodes 21 functions needed for the catabolism of 1,2-Pdl (31). Among the pdu genes, pduO has been shown to encode an ATP:co(I)rrinoid adenosyltransferase distinct from CobA (124, 125). The PduO-type enzyme is the most widely distributed type of corrinoid adenosyltransferase, with homologues found in archaea (229) and in eukaryotes ranging from yeast to humans (147). The serovar Typhimurium PduO enzyme is different from other PduO-type proteins in that its C-terminal 145 residues are not involved in catalysis (124); the function of this domain is currently unknown. The crystal structure of the serovar Typhimurium PduO enzyme has not been reported. However, structures of PduO-type enzymes from Thermoplasma acidophilum (apo PduO) (229), humans (an enzyme in a complex with ATP) (237), and Lactobacillus reuteri (an enzyme in a complex with ATP) (265) are available from protein databases. PduO-type enzymes bind ATP at a unique motif at the N terminus of the protein (237, 265). A better understanding of the active site of PduO-type enzymes awaits the resolution of the structures of the enzyme in complexes with its corrinoid and ATP substrates. There are some differences in corrinoid substrate specificity among PduO-type enzymes. For example, the serovar Typhimurium PduO enzyme is specific for Cbl, but the L. reuteri enzyme can also use Cbi as a substrate (265). An analysis of the PduO reaction products shows that, like CobA, PduO releases triphosphate as a by-product (124).
The eut operons of serovar Typhimurium and E. coli encode 16 functions needed for the utilization of ethanolamine as a carbon, nitrogen, and energy source (223, 224).
The eutT gene of the operons encodes a third class of ATP:co(I)rrinoid adenosyltransferases (44, 243). EutT is the least understood of the adenosyltransferases. However, several features distinguish EutT from CobA and PduO. First, EutT has substantially higher activity with ATP than with GTP. Second, EutT retains 80% of its activity when ADP substitutes for ATP, indicating important differences in the binding site for the nucleoside triphosphate substrate. Third, the by-products of the EutT reaction are not triphosphate but pyrophosphate and orthophosphate. Fourth, EutT activity is oxygen labile and is inactivated by the metal chelator bathophenanthroline; it is unclear why EutT is sensitive to this factor. Fifth, EutT is associated with the membrane, and detergent is needed to purify it (42, 44). Sixth, EutT binds ATP in an unconventional way since it lacks a P loop. Instead, EutT contains an HX7HX3CCX2C motif (residues conserved in the motif are shown in boldface) similar to the cytochrome oxidase CCX2C dicopper-binding site or to the 4Fe-4S cluster-binding motif recently identified in CbiX (34). Mutational analyses of EutT showed that when residue Cys79, Cys80, or Cys83 is changed to Ala, the resulting variant proteins lose >99% [EutT(C80A) and EutT(C83A)] or 80% [EutT(C79A)] of their activity; none of the proteins support the growth of a eutT strain on ethanolamine (42).
As defined above, cobamides are complete corrinoids that differ in the nature of their lower (Coα) ligand bases. The term cobalamin identifies the cobamide that contains DMB as the lower ligand. Serovar Typhimurium synthesizes at least three different cobamides as a function of oxygen in the environment. Under oxic conditions, serovar Typhimurium makes cobalamin (126, 133), but under anoxic conditions, serovar Typhimurium makes pseudo-B12 (adenylcobamide) or 2-methyladenylcobamide (134).
Insights into O2-Dependent DMB Synthesis Gained from Studies of Other Bacteria.
Studies of DMB synthesis in other bacteria established that aerobes derive DMB from flavin mononucleotide (203). Phenotypic analyses of Sinorhizobium meliloti DMB auxotrophs identified the bluB gene of Sinorhizobium meliloti as one encoding a function somehow involved in DMB synthesis (46, 296).
Nonenzymatic Synthesis of DMB.
At neutral pH and in the presence of O2, DMB is generated from 4,5-dimethylphenylenediamine and ribose 5-phosphate, and a mechanism for how this oxidative cascade occurs has been described previously (164). It is unclear, however, whether the mechanism of nonenzymatic DMB synthesis shares any steps with the BluB-triggered conversion of FMNH2 to DMB.
Single-Enzyme Conversion of FMNH2 to DMB.
In vitro evidence that the BluB protein from R. rubrum is necessary and sufficient for the O2-dependent conversion of FMNH2 to DMB has been reported (95). As has been observed with Sinorhizobium meliloti bluB strains, R. rubrum bluB strains cannot synthesize AdoCbl from Cbi unless DMB is present in the culture medium (95). However, the gene responsible for the synthesis of DMB in serovar Typhimurium or E. coli has not been identified. DMB pseudoauxotrophies displayed by cobT and cobC strains (49, 122, 185) are now understood and do not reflect a block in DMB synthesis in serovar Typhimurium (287, 313). To date, the isolation and characterization of bona fide DMB auxotrophs of serovar Typhimurium or E. coli have not been reported.
Lower-Ligand Synthesis in E.coli.
Studies of lower-ligand biosynthesis in E. coli have not been published. However, there is a report that describes the ability of E. coli to convert Cbi to Cbl or many other cobamides, depending on the base added to the culture medium (81). This result suggests that E. coli can assemble the nucleotide loop.
The NLA set of reactions are also known as the late steps of the AdoCbl biosynthetic pathway. The last three genes of the AdoCbl biosynthetic operon (cobUST; genome region, bp 2097303 to 2099685) encode NLA functions (184), with one additional NLA function gene (cobC) located outside the operon (genome region, bp 705072 to 705680) (185).
Phenotypic Analyses Provide a Physiological Framework to the Biochemistry Underpinning the NLA Pathway.
Most of the enzymatic activities associated with proteins of the NLA pathway have been known for several decades. Since the last edition of this work, enzymes have been studied in isolation, and structure-function studies of several of them have been performed. However, a physiological context for these functions was missing until recently. This important information was obtained through phenotypic analyses of serovar Typhimurium mutant strains unable to assemble the nucleotide loop. In addition to providing a physiological framework, these studies led to the discovery of new enzymes, pathways, and biosynthetic intermediates in bacteria and archaea.
CobU Is a Small Trifunctional Bacterial Enzyme Essential for Cobinamide Salvaging and De Novo AdoCbl Synthesis.
The activities of the CobU enzyme in extracts of propionic acid bacteria were first described three decades ago (221), but it took two decades before proteins with these activities were isolated from P. denitrificans (23) and serovar Typhimurium (183) and even longer before the physiological roles of these activities were elucidated (277).
Unlike de novo corrin ring biosynthesis, the NLA pathway (Fig. 8) is functional under anoxic and oxic conditions (122).
CobU is a small (<20-kDa) yet remarkably versatile enzyme. CobU has NTP:Ado-Cbi kinase and GTP:Ado-Cbi-P nucleotidyltransferase enzyme (EC 2.7.7.62 and EC 2.7.1.156) activities that synthesize Ado-cobinamide-GDP from AdoCbi and GTP (183). Under oxic conditions, CobU requires AdoCbi as the substrate, suggesting that the Ado moiety of AdoCbi is important for the recognition of AdoCbi under these conditions. Surprisingly, CobU can use nonadenosylated Cbi under anoxic conditions (182). Together, these observations suggest that CobU makes enough Cbi-GDP to satisfy the Cbl demands of a cell growing at a low rate, i.e., anoxically.
Some insights into the mechanism of the function of CobU are available. CobU guanylylates itself (in its third biochemical activity), forming a covalent phosphoramidate bond with the imidazole side chain of residue His46. Three-dimensional crystal structures of apo CobU and the CobU-GMP complex show a trimeric propeller-like form that undergoes a dramatic 10-Å conformational change upon guanylylation, bringing the GMP moiety closer to the P loop, where presumably the aminopropanol phosphate group of AdoCbi-P is bound (279, 280); the structure of CobU in a complex with its corrinoid substrate has not been reported. The GMP moiety bound to CobU is transferred to AdoCbi-P to yield the end product of the reaction (AdoCbi-GDP). The kinase reaction of CobU can use either ATP or GTP, but the transferase reaction is specific for GTP, and ATP inhibits it (278). The studies of CobU also showed that His46 is critical for kinase activity, suggesting that the enzyme uses a single active site for both reactions.
cobU alleles encoding CobU variant proteins with reduced kinase activity do not support the AdoCbl-dependent growth of serovar Typhimurium under oxic conditions, but they do support the de novo synthesis of the corrin ring under anoxic conditions (181). These observations, together with the discovery that the end product of the de novo corrin ring biosynthetic branch of the pathway is AdoCbi-P (40), led to the conclusion that the kinase activity is necessary for Cbi salvaging but not the de novo synthesis of AdoCbl. However, the guanylyltransferase activity of CobU is needed for Cbi salvaging and for de novo AdoCbl synthesis. These ideas were confirmed by the discovery of evolutionarily distinct archaeal enzymes that lack AdoCbi kinase activity but retain guanylyltransferase activity (277, 307). In turn, these discoveries led to the identification of a new pathway of Cbi salvaging in archaea (305, 306, 308).
CobT Is an Enzyme with a Striking Lack of Substrate Specificity That Affords Flexibility to the Pathway.
DMB is activated by CobT, a unique phosphoribosyltransferase (EC 2.4.2.21) that uses nicotinate mononucleotide (NaMN; a NAD+ precursor) or NAD+ as the donor of the phosphoribosyl moiety (165, 286).
CobT-like activity was first studied with cell-free extracts of Propionibacterium freudenreichii (formerly Propionibacterium shermanii) (86, 88) but has been isolated and biochemically characterized only from P. denitrificans and serovar Typhimurium (45, 285). What is unique about this enzyme is that the N-glycosidic bond in the reaction product is in the alpha configuration, a configuration that is retained in AdoCbl (Fig. 1).
Insights into the mechanism of catalysis used by CobT were obtained from extensive crystallographic analyses of the enzyme in complexes with NaMN, NaMN and DMB, and many other base substrates (52, 53, 55). The ability of this enzyme to use different base substrates explains the source of the diversity of cobamides in nature. The above-mentioned crystallographic studies also revealed that CobT cannot use phenol or p-cresol as a substrate, consistent with the idea that an evolutionarily distinct protein exists in Sporomusa ovata, a bacterium that synthesizes p-cresoyl and phenylcobamides (268, 269).
The CobB (Sirtuin) Protein Substitutes for CobT Function when the Requirement for Cbl Is Low.
The phenotype of serovar Typhimurium cobT strains is complex and misled researchers to conclude that CobT was involved in DMB synthesis (49). The Cob phenotype of cobT strains, however, was a valuable tool in the identification of the cobB gene, whose product compensates for the lack of CobT during AdoCbl synthesis from Cbi in the presence of DMB (286). The assignment of cob nomenclature to the cobB function was premature, since it is now known that CobB is not involved in AdoCbl synthesis. In fact, CobB is a NAD+-dependent protein deacetylase of the SIR2 family of proteins involved in the posttranslational control of protein activity in eukaryotes and prokaryotes (113, 258, 259, 287).
α-DAD, a New Intermediate of the NLA Pathway.
Findings from studies of the mechanism of catalysis of Sir2 proteins explain the serendipitous involvement of this mechanism in DMB activation (233, 250). The involvement of NAD+ in the CobB reaction led to the discovery that when CobT uses NAD+ as a substrate, the product of the reaction is α-DMB adenine dinucleotide (α-DAD), a previously unknown intermediate of the NLA pathway (165). The ability of CobT to use either NaMN or NAD+ provides physiological flexibility to microorganisms for the assembly of the nucleotide loop of AdoCbl. The enzyme responsible for cleaving α-DAD into AMP and α-ribazole-P has not been identified.
The CobS Enzyme Catalyzes the Penultimate Step of the NLA Pathway.
The CobS enzyme (EC 2.7.8.26) is an integral membrane protein (163), and until recently, CobS was thought to catalyze the last step of the AdoCbl biosynthetic pathway (163). However, a recent reassessment of the position of the CobS-catalyzed reaction within the NLA pathway indicates that CobS catalyzes the penultimate step, condensing AdoCbi-GDP (the product of the CobU enzyme) with α-ribazole-5'-P (the product of the CobT enzyme and of the unidentified enzyme that cleaves α-DAD into AMP and α-ribazole-5'-P) (313); the product of the CobS reaction is AdoCbl-5'-P. To date, CobS can be isolated only in small amounts. In spite of numerous efforts to increase its yield, the best conditions available give ~0.2 mg of CobS/liter of culture (312).
cobS overexpression correlates with the overproduction of PspA (phage shock protein A) (163), a non-DNA-binding protein that regulates its own expression. psp functions are conserved in many bacteria (e.g., E. coli, serovar Typhimurium, and Yersinia enterocolitica), and they are needed for survival in late stationary phase under conditions of high pH (66, 174). In E. coli and other bacteria, psp expression is activated by various extracytoplasmic stress stimuli, such as the mislocalization of protein into the cell envelope and alterations of the proton motive force (59).
Several questions regarding the physiology of the NLA pathway remain unanswered. For example, why is the CobS enzyme anchored to the cell membrane, and why does CobS overproduction elicit pspA gene expression? Interest in the answers to these questions is augmented by the fact that the pattern of localization of CobS to the inner membrane is shared by the CbiB enzyme, which catalyzes the last step of the de novo corrin ring biosynthetic pathway (308, 312).
What are the physiological advantages of localizing AdoCbl synthesis to the membrane? Whatever the answers are, the pattern of localization of these two proteins to the membrane is shared by all AdoCbl producers, judging by the sequence similarities of CobS and CbiB orthologues reported in databases. This shared feature of the pathway is certainly the result of strong selective pressure faced by all cobamide producers.
Elusive Role of the CobC Enzyme Brought to Light by the Phenotypic Analysis of the Intriguing Inability of cobC Mutant Strains To Salvage Cobyric Acid.
CobC-like activity in Propionibacterium freudenreichii (formerly Propionibacterium shermanii) was described previously (87), and its existence in P. denitrificans was mentioned (but not studied) (45), but only the serovar Typhimurium gene encoding the CobC enzyme has been isolated and overexpressed (185). In serovar Typhimurium, the cobC gene is transcribed divergently from cobD (bp 705777 to 706871). The CobC protein shows homology to phosphoglycerate mutase, the eukaryotic fructose 2,6-biphosphatase, and acid phosphatases. The Cob phenotypes of cobC strains are complex and depend on what incomplete corrinoid (e.g., Cby or Cbi) is provided in the medium and whether the environment is devoid of oxygen or not (185, 313). In fact, under oxic growth conditions that demand Cbi salvaging, the effect of the lack of CobC on AdoCbl synthesis is observed only in a cobC cobT double mutant strain (185).
Initial in vitro studies of CobC phosphatase (EC 3.1.3.73) activity showed that the enzyme used α-ribazole-P as a substrate, suggesting that CobS was the last step of the pathway. However, subsequent studies showed that AdoCbl-P was also a substrate of CobC, leaving unresolved the question of what was the true physiological substrate of CobC. Recent in vivo and in vitro studies of cobyric acid salvaging established that CobC catalyzes the last step of the pathway, that AdoCbl-5'-P is the physiological substrate of CobC, and that α-ribazole is probably not an intermediate of the pathway (313).
NLA in E.coli.
As mentioned above, it has been known for >5 decades that E. coli can synthesize AdoCbl when the AdoCbl precursor Cbi is added to the culture medium (80). However, it was not until 1995 that evidence for the lack of de novo corrin ring biosynthetic functions in this bacterium was obtained (145). That E. coli cannot synthesize the corrin ring de novo was confirmed when the sequence of the E. coli genome was reported (28). The genome of this bacterium, however, does include cobUST homologues that are functional, allowing it to salvage Cbi from its environment (145). Unlike that of the cobUST genes of serovar Typhimurium, the expression of the cobUST genes of E. coli is induced by Cbi but not by 1,2-Pdl (145).
The expression of the AdoCbl biosynthetic operon is complex and is modulated at different levels. At the transcriptional level, a sensor response regulator protein activates the transcription of the operon in response to 1,2-Pdl in the environment. Global regulatory proteins control the transcription of the operon as a function of the redox state and the quality of the carbon source in the environment. Other functions affect the expression of the operon, but it is unclear whether their effects are direct or indirect. Translation initiation control of the AdoCbl biosynthetic genes is exerted by an AdoCbl-responsive riboswitch.
The 1,2-Pdl dependent regulation of AdoCbl synthesis has been reviewed previously (71), and no new information has been published since then. Highlights of this mode of control are presented in Fig. 9.
The discovery that 1,2-Pdl induces the expression of the AdoCbl biosynthetic operon under oxic and anoxic conditions was exciting and triggered a great deal of in vivo and in vitro experimentation (218). Under oxic or anoxic conditions and with 1,2-Pdl in the medium, the expression of the cbi genes is increased between 30- and 43-fold compared to that in the absence of 1,2-Pdl, while cobUST expression is increased ~1 order of magnitude. Because the transcription of the pdu operon is also positively affected by 1,2-Pdl (121), the cbi and pdu operons comprise a regulon.
The PocR Protein Is a Transcriptional Regulator That Coregulates the Expression of the cbi and pdu Operons.
Mutations abolishing the effect of 1,2-Pdl were located in the poc locus situated between cbiA and pduA (30, 218). DNA sequencing of poc identified two open reading frames, namely, pocR and pduF (50, 226). PocR is a DNA-binding protein (216) and is a member of the AraC family of transcriptional activators (50, 191, 281); PocR mediates the 1,2-Pdl effect. In vitro studies of how PocR activates the transcription of the cbi genes have been reported (216). Two PocR-binding sites located immediately in the 5' direction from the cbiA gene were identified, and in between the PocR sites there is an integration host factor (IHF)-binding site, presumably needed to bend DNA and allow PocR-dependent activation of the cbi genes. The role of IHF in cob-pdu regulon expression is highlighted by the inability of himB mutant strains to grow on 1,2-Pdl (217). The oligomeric state of PocR has not been determined, but by analogy to other AraC-like activators, it may function as a dimer.
The expression of the pocR gene is regulated aerobically and anaerobically. Aerobically, pocR gene expression is induced about threefold in response to 1,2-Pdl. This effect is mediated by PocR and requires the cyclic AMP (cAMP) receptor protein (CRP)-cAMP system (30). Anaerobically, pocR gene expression is also induced about threefold in response to 1,2-Pdl (30). Under the conditions used by Bobik et al. (30), both the CRP-cAMP and ArcAB systems were involved in controlling cbi operon expression.
The PduF Protein Is a 1,2-Pdl Transport Facilitator.
PduF and the glycerol facilitator GlpF proteins are homologues, showing 65% identity and 83% similarity at the amino acid level (50). The expression of the pduF gene is controlled from two different promoters activated by the PocR–1,2-Pdl complex (48). Detailed studies of the function of PduF have not been reported.
The expression of the AdoCbl biosynthetic operon is induced when cells are grown under anaerobic conditions (72). Higher levels of induction are correlated with the presence of an external electron acceptor having a lower midpoint potential; this phenomenon is referred to as redox regulation (3, 72). Studies of the involvement of the ArcAB two-component regulatory system (117, 167) suggest that the redox environment, not the absence of oxygen, inside the cell is critical for cbi transcription (1, 2). Although putative binding sites for ArcA-P appear to exist in the 5' direction from the pduF gene, direct in vitro proof of ArcA-P binding to this region has not been reported.
The involvement of the global regulatory CRP-cAMP system was reported in early regulation studies of the AdoCbl biosynthetic operon (72). Further studies of the involvement of the CRP-cAMP system led to the proposal that the major role of AdoCbl in serovar Typhimurium is in the catabolism of carbon sources, specifically 1,2-Pdl (1).
Early studies of regulation of cbi/cob operon expression reported Cbl to be a negative effector of this expression (72), but the connection between AdoCbl and the posttranscriptional control of cbi/cob expression was not made until later, by A. A. Richter-Dahlfors and D. I. Andersson (204). These researchers performed detailed, elegant experiments to show that the posttranscriptional mechanism requires sequences within the leader and the cbiA gene, the first gene of the operon (205). Andersson and S. Ravnum also demonstrated that repression by Cbl requires the proper folding of a hairpin in the RNA for the sequestration of the ribosome-binding site blocking translation initiation as a function of the end product of the pathway, i.e., AdoCbl (201). Ravnum and Andersson extended these observations to the regulatory region of the gene encoding the outer membrane corrinoid transport protein BtuB (202).
In E. coli, the correlation between AdoCbl and translational repression of the btuB gene was also established early by R. Kadner and coworkers, arriving at the same conclusion regarding the mechanism of repression via the sequestration of the ribosome-binding site of btuB mRNA (159, 160, 161, 179).
The term "riboswitch" was coined to refer to this type of control of gene expression, in which the 5' untranslated regions of some genes modulate mRNA translation as a function of changing concentrations of metabolites (178). A. Nahvi and coworkers have further advanced our understanding of the function of the cbi and btuB riboswitches and have shown that this type of genetic control of AdoCbl biosynthesis and transport genes is widespread among gram-positive and gram-negative prokaryotes (177).
It was reported previously that glutathione (GSH)-deficient (gshA) strains of serovar Typhimurium cannot grow on 1,2-Pdl under aerobic conditions (220). The reported studies showed that the lack of GSH clearly affects cbi and pdu expression, but questions remain as to whether one or more of the proteins controlling the expression of the regulon are affected by the lack of GSH. Under anoxic conditions, the absence of GSH does not affect the expression of the regulon, suggesting that a redox role for GSH is plausible. Although some ideas regarding a redox role for GSH have been proposed (220), this very interesting observation needs to be further investigated.
The expression of the eut operon is controlled by the EutR protein (222), an AraC/XylS-type transcriptional activator that requires AdoCbl and ethanolamine as coactivators (44, 244). In vivo genetic data show that EutR also controls its own synthesis (222). At present, the mechanistic details of EutR function are lacking. Reports of a role for GSH in ethanolamine utilization ruled out an effect on EutR function. Instead, it was suggested that in the absence of GSH, ethanolamine ammonia-lyase loses >90% of its activity; the reasons for such a loss of activity are not known (220).
The regulation of the cobA and the cobD genes has been examined previously. None of the effectors that regulate the expression of the main AdoCbl biosynthetic operon (1,2-Pdl, redox, a carbon source, and AdoCbl) have an effect on cobA or cobD transcription (73, 94). Studies of the regulation of the cobC and cobD genes are of interest since these genes neighbor each other and are divergently transcribed and the promoters driving their expression are most likely embedded in each other’s coding sequences (40).
The following factors affect the expression of the AdoCbl operon.
IHF. Serovar Typhimurium ihf strains do not grow on 1,2-Pdl (217). Five putative IHF-binding sites are present in the cbiA-pduA regulatory region (144), but detailed in vitro analyses of the interaction of IHF with most of these sites are lacking
RpoS sigma factor. Increased levels of the RpoS sigma factor eliminate the positive effect of 1,2-Pdl on cbi-pdu regulon expression (215). Here again, details of how RpoS levels affect AdoCbl synthesis are missing
Carbon storage regulator (CsrA). The CsrA protein was first discovered during studies of functions that regulate glycogen biosynthesis in E. coli (214). The CsrABC system is widespread among bacteria, with CsrA modulating gene expression posttranscriptionally by controlling the decay of specific mRNAs and CsrB and CsrC noncoding RNAs antagonizing CsrA activity (142, 172, 212, 213). Details of the CsrA-mRNA interactions are beginning to emerge, and the interpretation of the data is facilitated by the availability of the crystal structure of CsrA (206). S. D. Lawhon and coworkers discovered that in a csrA strain of serovar Typhimurium, the expression of genes involved in AdoCbl biosynthesis and use is substantially reduced compared to that in the wild type (143). It remains unclear, however, how CsrA exerts its effect on cbi/cob, eut, and pdu expression
As mentioned above, serovar Typhimurium and E. coli use ethanolamine as a source of carbon, nitrogen, and energy. In addition, and unlike E. coli, serovar Typhimurium can also grow on 1,2-Pdl as the sole source of carbon and energy. The biochemistry underpinning these catabolic pathways includes AdoCbl-dependent enzymes catalyzing the dehydration of 1,2-Pdl or the deamination of ethanolamine. In both cases, the product of the reaction is an aldehyde (acetaldehyde or propionaldehyde), which is oxidized by a CoA-dependent dehydrogenase, yielding the corresponding acyl-CoA derivative. Ethanolamine and 1,2-Pdl catabolism processes require functions encoded by two large operons, the 16-gene ethanolamine utilization operon (eutSPQTDMNEJGHABCLK) (138) and the 21-gene 1,2-Pdl utilization operon (pduABCDEGHJKLMNOPQSTUVWX) (31), respectively.
The catabolism of these poor carbon sources requires the assembly of multiprotein structures similar to the carboxysome found in many bacteria that use ribulose bisphosphate carboxylase/oxygenase (RuBisCO) to fix carbon dioxide (131, 180, 235, 247, 252, 311). Although the term "carboxysome-like" has been used to describe the structures assembled for the catabolism of ethanolamine and 1,2-Pdl, the term "metabolosome" was proposed to reflect the involvement of these structures in metabolism that does not involve carbon dioxide (36).
Seven of the eut genes encode proteins with enzyme (EutTDABC) or permease (EutH) activities (35, 42, 44, 75, 175, 190, 243). Putative enzymatic activities have been proposed on the basis of homology for EutE and EutG, but these assignments of function are not supported by in vitro data; EutJ may be a chaperone (190). There is no putative function for EutP or EutQ, and EutSMNLK show homology to shell proteins found in the carboxysome (266).
Ethanolamine catabolism in the absence of the metabolosome has been reported (36). In fact, serovar Typhimurium grows very well on ethanolamine in the absence of the entire operon as long as the eutBC genes (encoding ethanolamine ammonia-lyase) are provided on a plasmid and GSH is present in the medium (36). The effect of GSH on ethanolamine catabolism was reported over a decade ago (220), is not understood, and deserves further investigation.
The reported requirement for DNA polymerase I (PolA) during the growth of serovar Typhimurium on ethanolamine is intriguing (219). Whether the effect of the lack of PolA is direct or indirect remains to be determined.
The majority of research on the components of the Pdl utilization (pdu) system has been carried out by Tom Bobik’s group (29). The sequencing of the complete pdu locus revealed that it contains 23 genes, 21 of which are organized into an operon (31). The genes include, as expected, those encoding enzymes associated with the catabolism of 1,2-Pdl, including the B12-dependent diol dehydratase (32). However, what was surprising was the identification of genes that encode proteins with high degrees of similarity to those associated with polyhedral organelle formation (31). Initially, polyhedral organelles were observed in cyanobacteria and were termed carboxysomes (90, 248). The carboxysome consists of a metabolic enzyme encased within a multiprotein shell, similar to, though quite distinct from, a viral capsid. In the case of cyanobacteria and thiobacilli, this organelle is involved in carbon dioxide concentration and fixation (hence the term carboxysome) via the enzymes carbonic anhydrase and RuBisCO. In serovar Typhimurium, it was found that the pdu-encoded polyhedra form during both aerobic and anaerobic growth on Pdl (31). It was also shown that the diol dehydratase is associated with the metabolosome, indicating that the metabolosomes are associated directly with Pdl metabolism.
The metabolic advantage of the carboxysome is thought to lie in the improvement of CO2 fixation via the Calvin cycle by enhancing the activity of RuBisCO at low CO2 concentrations. It is not clear why Pdl (and, for that matter, ethanolamine) is metabolized within such microcompartments, although it has been hypothesized that since their breakdown proceeds via aldehydes, it is to protect the cell from the harmful accumulation of such an intermediate (101, 219). It has also been suggested that as the aldehydes are volatile, the organelle may act to conserve these metabolites, for example, by providing a low-pH environment (190). There is significant interest in how these macromolecular assemblies form and how they compartmentalize their respective metabolic enzymes, substrates, cofactors, and products.
For Pdl metabolism, 23 gene products are known to be involved, including metabolic enzymes such as the B12-dependent diol dehydratase (PduC, PduD, and PduE) and the aldehyde dehydrogenase (PduP), as well as the shell proteins and transcriptional regulators (31). Significantly, the shell proteins of ethanolamine utilization and Pdl metabolism display some sequence similarity among themselves and to the carboxysome shell proteins, indicating a common evolutionary ancestry (29). Thus, PduA, PduJ, PduK, PduT, PduB, and PduU and EutS, EutM, EutN, EutL, and EutK all display various degrees of similarity (between 15 and 50% identity) to the carboxysome shell proteins CsoS1, CcmK, and CcmO. Consequently, there is likely to be some degree of structure conservation among these organelles. Significantly, recent X-ray structure work has led to the identification of the carboxysome shell protein CcmK, or actually, two variants termed CcmK2 and CcmK4, which are comparatively small proteins, each consisting of a total of about 100 amino acid residues (136). These monomers fit together to form a hexamer with a positively charged central pore. The hexamers appear to link together into sheets that may serve as surfaces for the organelle. The pore may act as an entry and exit site for metabolites, although the crystal structure indicates that the pore is likely to be specific for negatively charged substrates and products of RuBisCO. The crystal structure of the hexamers is the only known structure of a bacterial organelle protein. There is thus a great deal still to learn about how the various shell proteins come together to form the macromolecular complex. Moreover, there are many interesting questions relating to how the metabolic enzymes are interned and arranged within the organelle, as well as how the substrates, coenzymes, and products make their way into and out of the structure.
The protein content of the isolated pdu metabolosome was studied after the isolation of the organelle by density gradient centrifugation (100). The protein components of the complex were separated by two-dimensional electrophoresis, and the various proteins were identified. This analysis revealed that the organelles are composed of at least 13 pdu-encoded gene products, including PduA, PduB, PduC, PduD, PduE, PduG, PduH, PduK, PduO, PduP, PduT, and PduU. Moreover, there appeared to be at least one unidentified protein, and PduB was also detected in a truncated form, missing the N-terminal 37 amino acids. Of these proteins, PduA, PduB, PduJ, PduK, PduT, and PduU have some degree of similarity to the carboxysome shell proteins (31), whereas PduC, PduD, and PduE are the diol dehydratase subunits, PduG and PduH are the diol dehydratase reactivation factors, PduO is an adenosyltransferase for adenosylcobalamin formation (124, 125), and PduP is a CoA-dependent propionaldehyde dehydrogenase (146). The findings of this organelle-proteomics study clearly demonstrate that the metabolosome is a multisubunit complex containing a mixture of structural (shell) proteins and metabolic enzymes.
Of the Pdu shell proteins, PduA is the only one to be studied in any detail (101). In that analysis, nonpolar mutations in PduA resulted in the loss of the formation of the metabolosomes whereas the overproduction of PduA gave rise to the appearance of aberrant rod-shaped structures. Antibodies raised against PduA were found to localize to the shell of the organelle. All the evidence suggests that PduA is a structural protein whose correct ratio is important in the formation of the metabolsome.
The metabolosome is used to metabolize 1,2-Pdl. The analysis of the composition of the organelle in comparison to that of proteins found encoded in the pdu operon has allowed a model for the functioning of this organelle to be proposed (29). Pdl enters the metabolosome, formed from a number of structural shell proteins including PduA, PduB, PduJ, PduK, PduT, and PduU (32), where it is acted upon by the diol dehyratase (PduC, PduD, and PduE) (32). The enzyme requires adenosylcobalamin as a coenzyme, and as cobalamin is taken up into the organelle, so the corrin is adenosylated by the action of PduO (124). The adenosylation of cobalamin requires the reduction of the central cobalt ion to the Co(I) form, and this activity has been attributed to PduS (228). Although the localization of PduS has not been determined, the recombinant protein has been produced and the enzyme has been shown to have both Co(III)-to-Co(II) and Co(II)-to-Co(I) activities. Evidence has also been presented to suggest that PduO and PduS may interact to channel the highly nucleophilic Co(I) species (228). The catalytic cycle of the diol dehydratase occasionally undergoes suicide inactivation, and the complex has to be reactivated. This process is accomplished by the reactivation factors PduG and PduH (31, 283). The dehydration of Pdl results in the production of propionaldehyde, which is then dismuted into 1-propanol and propionyl-CoA (29). The transformation of propionaldehyde into 1-propanol is catalyzed by PduQ in a reaction that requires NADH and is likely to occur outside of the metabolosome (31, 100). The formation of propionyl-CoA from propionaldehyde is likely to occur within the organelle and is catalyzed by PduP, a CoA-acylating propionaldehyde dehydrogenase (146). Localization studies have shown that PduP is located within the organelle, and recombination studies have confirmed the activity of the enzyme. Propionyl-CoA is then thought to diffuse out of the organelle, where it is acted upon by PduL, a phosphotransacylase that generates propionyl phosphate (155). The final stage in the process is catalyzed by PduW, which is a propionate kinase (29, 31, 186).
In spite of the substantial progress achieved in the last two decades, there are several lofty goals that need to be met before we have a solid understanding of AdoCbl biosynthesis and use in serovar Typhimurium and E. coli. Some of these goals are as follows:
Identification of the gene encoding the FMNH2-dependent DMB synthase and elucidation of its mechanism of catalysis
Structure-function analyses of many of the Cbi and Cob enzymes
Analyses of regulation of the expression of genes outside the cbi/cob operon
Achievement of a better understanding of the physiological reasons that drive the association of AdoCbl biosynthetic enzymes with the cell membrane
Study of protein-protein interactions and what appear to be large, multiprotein complexes
Structure-function analyses of the Eut and Pdu metabolosomes
Elucidation of the functions of all the proteins encoded by the eut and pdu operons
Determination of the role the methylmalonyl-CoA mutase enzyme in E. coli physiology
These and other goals will pose great challenges to researchers in this field of prokaryotic physiology. The answers to these questions will undoubtedly be very exciting and will advance our understanding of the reasons for making such a complex coenzyme.
This work was supported by PHS grant R01-GM40313 to J.C.E.-S. and by funding from the Biotechnology and Biological Sciences Research Council (BBSRC) to M.J.W.
References
1. Ailion, M., T. A. Bobik, and J. R. Roth. 1993. Two global regulatory systems (Crp and Arc) control the cobalamin/propanediol regulon of Salmonella typhimurium. J. Bacteriol. 175:7200–7208.[PubMed]
2. Andersson, D. I. 1992. Involvement of the Arc system in redox regulation of the Cob operon in Salmonella typhimurium. Mol. Microbiol. 6:1491–1494. [CrossRef]
3. Andersson, D. I., and J. R. Roth. 1989. Redox regulation of the genes for cobinamide biosynthesis in Salmonella typhimurium. J. Bacteriol. 171:6734–6739.[PubMed]
3a. Anzai, Y., H. Kim, J. Y. Park, H. Wakabayashi, and H. Oyaizu. 2000. Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int. J. Syst, Evol. Microbiol. 50:1563–1589.
4. Bandarian, V., M. L. Ludwig, and R. G. Matthews. 2003. Factors modulating conformational equilibria in large modular proteins: a case study with cobalamin-dependent methionine synthase. Proc. Natl. Acad. Sci. USA 100:8156–81563.[PubMed] [CrossRef]
5. Bandarian, V., and R. G. Matthews. 2004. Measurement of energetics of conformational change in cobalamin-dependent methionine synthase. Methods Enzymol. 380:152–169.[PubMed] [CrossRef]
6. Bandarian, V., and R. G. Matthews. 2001. Quantitation of rate enhancements attained by the binding of cobalamin to methionine synthase. Biochemistry 40:5056–5064.[PubMed] [CrossRef]
7. Bandarian, V., K. A. Pattridge, B. W. Lennon, D. P. Huddler, R. G. Matthews, and M. L. Ludwig. 2002. Domain alternation switches B(12)-dependent methionine synthase to the activation conformation. Nat. Struct. Biol. 9:53–56.[PubMed] [CrossRef]
8. Bandarian, V., and G. H. Reed. 1999. Ethanolamine ammonia-lyase, p. 811–833. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York, NY.
9. Banerjee, R., and S. Chowdhury. 1999. Methylmalonyl-CoA mutase, p. 707–729. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York, NY.
10. Banerjee, R. V., S. R. Harder, S. W. Ragsdale, and R. G. Matthews. 1990. Mechanism of reductive activation of cobalamin-dependent methionine synthase: an electron paramagnetic resonance spectroelectrochemical study. Biochemistry 29:1129–1135.[PubMed] [CrossRef]
11. Banerjee, R. V., and R. G. Matthews. 1990. Cobalamin-dependent methionine synthase. FASEB J. 4:1450–1459.[PubMed]
12. Bartosinsky, B., B. Zagalak, and J. Pawelkiewicz. 1967. The route of vitamin B12 biosynthesis in Propionibacterium shermanii. Biochim. Biophys. Acta 136:581–584.[PubMed]
13. Bassford, P. J., Jr., and R. J. Kadner. 1977. Genetic analysis of components involved in vitamin B12 uptake in Escherichia coli. J. Bacteriol. 132:796–805.[PubMed]
14. Battersby, A. R. 1994. How nature builds the pigments of life: the conquest of vitamin B12. Science 264:1551–1557.[PubMed] [CrossRef]
15. Battersby, A. R. 1994. New intermediates in the B12 pathway, p. 267–279. In D. J. Chadwick and K. Ackrill (ed.), CIBA Foundation Symposium on the Biosynthesis of the Tetrapyrrole Pigments. John Wiley & Sons, New York, NY.
16. Battersby, A. R. 2000. Tetrapyrroles: the pigments of life. Nat. Prod. Rep. 17:507–526.[PubMed] [CrossRef]
17. Battersby, A. R., C. J. Fookes, G. W. Matcham, and E. McDonald. 1980. Biosynthesis of the pigments of life: formation of the macrocycle. Nature 285:17–21.[PubMed] [CrossRef]
18. Bauer, C. B., M. V. Fonseca, H. M. Holden, J. B. Thoden, T. B. Thompson, J. C. Escalante-Semerena, and I. Rayment. 2001. Three-dimensional structure of ATP:corrinoid adenosyltransferase from Salmonella typhimurium in its free state, complexed with MgATP, or complexed with hydroxycobalamin and MgATP. Biochemistry 40:361–374.[PubMed] [CrossRef]
19. Beale, S. I., and P. A. Castelfranco. 1973. 14C incorporation from exogenous compounds into δ-aminolevulinic acid by greening cucumber cotyledons. Biochem. Biophys. Res. Commun. 52:143–149.[PubMed] [CrossRef]
20. Blackwell, C. M., F. A. Scarlett, and J. M. Turner. 1976. Ethanolamine catabolism by bacteria, including Escherichia coli. Biochem. Soc. Trans. 4:495–497.[PubMed]
21. Blackwell, C. M., and J. M. Turner. 1978. Microbial metabolism of amino alcohols. Formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli. Biochem. J. 176:751–757.[PubMed]
22. Blanche, F., B. Cameron, J. Crouzet, L. Debussche, D. Thibaut, M. Vuilhorgne, F. J. Leeper, and A. R. Battersby. 1995. Vitamin B12: how the problem of its biosynthesis was solved. Angew. Chem. (International ed.) 34:383–411. [CrossRef]
23. Blanche, F., L. Debussche, A. Famechon, D. Thibaut, B. Cameron, and J. Crouzet. 1991. A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities. J. Bacteriol. 173:6052–6057.[PubMed]
24. Blanche, F., L. Debussche, D. Thibaut, J. Crouzet, and B. Cameron. 1989. Purification and characterization of S-adenosyl-l-methionine:uroporphyrinogen III methyltransferase from Pseudomonas denitrificans. J. Bacteriol. 171:4222–4231.[PubMed]
25. Blanche, F., A. Famechon, D. Thibaut, L. Debussche, B. Cameron, and J. Crouzet. 1992. Biosynthesis of vitamin B12 in Pseudomonas denitrificans: the biosynthetic sequence from precorrin-6y to precorrin-8x is catalyzed by the cobL gene product. J. Bacteriol. 174:1050–1052.[PubMed]
26. Blanche, F., D. Thibaut, L. Debussche, R. Hertle, F. Zipfel, and G. Muller. 1993. Parallels and decisive differences in vitamin B12 biosynthesis. Angew. Chem. (International ed.) 32:1651–1653. [CrossRef]
27. Blanche, F., D. Thibaut, A. Famechon, L. Debussche, B. Cameron, and J. Crouzet. 1992. Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene. J. Bacteriol. 174:1036–1042.[PubMed]
28. Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453–1474.[PubMed] [CrossRef]
29. Bobik, T. A. 2006. Polyhedral organelles compartmenting bacterial metabolic processes. Appl. Microbiol. Biotechnol. 70:517–525.[PubMed] [CrossRef]
30. Bobik, T. A., M. Ailion, and J. R. Roth. 1992. A single regulatory gene integrates control of vitamin B12 synthesis and propanediol degradation. J. Bacteriol. 174:2253–2266.[PubMed]
31. Bobik, T. A., G. D. Havemann, R. J. Busch, D. S. Williams, and H. C. Aldrich. 1999. The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 includes genes necessary for formation of polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degradation. J. Bacteriol. 181:5967–5975.[PubMed]
32. Bobik, T. A., Y. Xu, R. M. Jeter, K. E. Otto, and J. R. Roth. 1997. Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase. J. Bacteriol. 179:6633–6639.[PubMed]
33. Bouquiere, J. P., J. L. Finney, M. S. Lehmann, P. F. Lindley, and H. F. J. Savage. 1993. High-resolution neutron study of vitamin B12 coenzyme at 15K: structure analysis and comparison with the structure at 279K. Acta Crystallogr. B49:79–89.
34. Brindley, A. A., E. Raux, H. K. Leech, H. L. Schubert, and M. J. Warren. 2003. A story of chelatase evolution: identification and characterization of a small 13–15-kDa "ancestral" cobaltochelatase (CbiXS) in the archaea. J. Biol. Chem. 278:22388–22395.[PubMed] [CrossRef]
35. Brinsmade, S. R., and J. C. Escalante-Semerena. 2004. The eutD gene of Salmonella enterica encodes a protein with phosphotransacetylase enzyme activity. J. Bacteriol. 186:1890–1892.[PubMed] [CrossRef]
36. Brinsmade, S. R., T. Paldon, and J. C. Escalante-Semerena. 2005. Minimal functions and physiological conditions required for growth of Salmonella enterica on ethanolamine in the absence of the metabolosome. J. Bacteriol. 187:8039–8046.[PubMed] [CrossRef]
37. Brown, E. D. 2005. Conserved P-loop GTPases of unknown function in bacteria: an emerging and vital ensemble in bacterial physiology. Biochem. Cell Biol. 83:738–746.[PubMed] [CrossRef]
38. Brown, K. L. 2005. Chemistry and enzymology of vitamin B12. Chem. Rev. 105:2075–2149.[PubMed] [CrossRef]
39. Brown, K. L. 1999. NMR spectroscopy of B12, p. 197–237. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York, NY.
40. Brushaber, K. R., G. A. O’Toole, and J. C. Escalante-Semerena. 1998. CobD, a novel enzyme with l-threonine-O-3-phosphate decarboxylase activity, is responsible for the synthesis of (R)-1-amino-2-propanol O-2-phosphate, a proposed new intermediate in cobalamin biosynthesis in Salmonella typhimurium LT2. J. Biol. Chem. 273:2684–2691.[PubMed] [CrossRef]
41. Buan, N. R., and J. C. Escalante-Semerena. 2005. Computer-assisted docking of flavodoxin with the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme reveals residues critical for protein-protein interactions but not for catalysis. J. Biol. Chem. 280:40948–40956.[PubMed] [CrossRef]
42. Buan, N. R., and J. C. Escalante-Semerena. 2006. Purification and initial biochemical characterization of ATP:cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica. J. Biol. Chem. 281:16971–16977.[PubMed] [CrossRef]
43. Buan, N. R., K. Rehfeld, and J. C. Escalante-Semerena. 2006. Studies of the CobA-type ATP:co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Gö1. J. Bacteriol. 188:3543–3550.[PubMed] [CrossRef]
44. Buan, N. R., S. J. Suh, and J. C. Escalante-Semerena. 2004. The eutT gene of Salmonella enterica encodes an oxygen-labile, metal-containing ATP:corrinoid adenosyltransferase enzyme. J. Bacteriol. 186:5708–5714.[PubMed] [CrossRef]
45. Cameron, B., F. Blanche, M. C. Rouyez, D. Bisch, A. Famechon, M. Couder, L. Cauchois, D. Thibaut, L. Debussche, and J. Crouzet. 1991. Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide:dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5'-phosphate) synthase. J. Bacteriol. 173:6066–6073.[PubMed]
46. Campbell, G. R., M. E. Taga, K. Mistry, J. Lloret, P. J. Anderson, J. R. Roth, and G. C. Walker. 2006. Sinorhizobium meliloti bluB is necessary for production of 5,6-dimethylbenzimidazole, the lower ligand of B12. Proc. Natl. Acad. Sci. USA 103:4634–4369.[PubMed] [CrossRef]
47. Chang, G. W., and J. T. Chang. 1975. Evidence for the B12-dependent enzyme ethanolamine deaminase in Salmonella. Nature 254:150–151.[PubMed] [CrossRef]
48. Chen, P., M. Ailion, T. Bobik, G. Stormo, and J. Roth. 1995. Five promoters integrate control of the cob/pdu regulon in Salmonella typhimurium. J. Bacteriol. 177:5401–5410.[PubMed]
49. Chen, P., M. Ailion, N. Weyand, and J. Roth. 1995. The end of the cob operon: evidence that the last gene (cobT) catalyzes synthesis of the lower ligand of vitamin B12, dimethylbenzimidazole. J. Bacteriol. 177:1461–1419.[PubMed]
50. Chen, P., D. I. Andersson, and J. R. Roth. 1994. The control region of the pdu/cob regulon in Salmonella typhimurium. J. Bacteriol. 176:5474–5482.[PubMed]
51. Cheong, C. G., C. B. Bauer, K. R. Brushaber, J. C. Escalante-Semerena, and I. Rayment. 2002. Three-dimensional structure of the l-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica. Biochemistry 41:4798–4808.[PubMed] [CrossRef]
52. Cheong, C. G., J. C. Escalante-Semerena, and I. Rayment. 2002. Capture of a labile substrate by expulsion of water molecules from the active site of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica. J. Biol. Chem. 277:41120–41127.[PubMed] [CrossRef]
53. Cheong, C. G., J. C. Escalante-Semerena, and I. Rayment. 2001. Structural investigation of the biosynthesis of alternative lower ligands for cobamides by nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase from Salmonella enterica. J. Biol. Chem. 276:37612–37620.[PubMed] [CrossRef]
54. Cheong, C. G., J. C. Escalante-Semerena, and I. Rayment. 2002. Structural studies of the l-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica: the apo, substrate, and product-aldimine complexes. Biochemistry 41:9079–9089.[PubMed] [CrossRef]
55. Cheong, C. G., J. C. Escalante-Semerena, and I. Rayment. 1999. The three-dimensional structures of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium complexed with 5,6-dimethybenzimidazole and its reaction products determined to 1.9 Å resolution. Biochemistry 38:16125–16135.[PubMed] [CrossRef]
56. Croft, M. T., A. D. Lawrence, E. Raux-Deery, M. J. Warren, and A. G. Smith. 2005. Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature 438:90–93.[PubMed] [CrossRef]
57. Croft, M. T., M. J. Warren, and A. G. Smith. 2006. Algae need their vitamins. Eukaryot. Cell 5:1175–1183.[PubMed] [CrossRef]
58. Crouzet, J., L. Cauchois, F. Blanche, L. Debussche, D. Thibaut, M. C. Rouyez, S. Rigault, J. F. Mayaux, and B. Cameron. 1990. Nucleotide sequence of a Pseudomonas denitrificans 5.4-kilobase DNA fragment containing five cob genes and identification of structural genes encoding S-adenosyl-l-methionine:uroporphyrinogen III methyltransferase and cobyrinic acid a,c-diamide synthase. J. Bacteriol. 172:5968–5979.[PubMed]
59. Darwin, A. J. 2005. The phage-shock-protein response. Mol. Microbiol. 57:621–628.[PubMed] [CrossRef]
60. Debussche, L., D. Thibaut, B. Cameron, J. Crouzet, and F. Blanche. 1993. Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans. J. Bacteriol. 175:7430–7440.[PubMed]
61. DiMarco, A. A., T. A. Bobik, and R. S. Wolfe. 1990. Unusual coenzymes of methanogenesis. Annu. Rev. Biochem. 59:355–94.[PubMed] [CrossRef]
62. Dorweiler, J. S., R. G. Finke, and R. G. Matthews. 2003. Cobalamin-dependent methionine synthase: probing the role of the axial base in catalysis of methyl transfer between methyltetrahydrofolate and exogenous cob(I)alamin or cob(I)inamide. Biochemistry 42:14653–14662.[PubMed] [CrossRef]
63. Dorweiler, J. S., R. G. Matthews, and R. G. Finke. 2002. Providing a chemical basis toward understanding the histidine base-on motif of methylcobalamin-dependent methionine synthase: an improved purification of methylcobinamide, plus thermodynamic studies of methylcobinamide binding exogenous imidazole and pyridine bases. Inorg. Chem. 41:6217–6224.[PubMed] [CrossRef]
64. Drennan, C. L., S. Huang, J. T. Drummond, R. G. Matthews, and M. L. Ludwig. 1994. How a protein binds B12: a 3.0 Å X-ray structure of B12-binding domains of methionine synthase. Science 266:1669–1674.[PubMed] [CrossRef]
65. Drummond, J. T., and R. G. Matthews. 1993. Cobalamin-dependent and cobalamin-independent methionine synthases in Escherichia coli: two solutions to the same chemical problem. Adv. Exp. Med. Biol. 338:687–692.[PubMed]
66. Dworkin, J., G. Jovanovic, and P. Model. 2000. The PspA protein of Escherichia coli is a negative regulator of σ54-dependent transcription. J. Bacteriol. 182:311–319.[PubMed] [CrossRef]
67. Elliott, T., Y. J. Avissar, G. E. Rhie, and S. I. Beale. 1990. Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase. J. Bacteriol. 172:7071–7084.[PubMed]
68. Elliott, T., and J. R. Roth. 1989. Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis. Mol. Gen. Genet. 216:303–314.[PubMed] [CrossRef]
69. Erskine, P. T., L. Coates, R. Newbold, A. A. Brindley, F. Stauffer, S. P. Wood, M. J. Warren, J. B. Cooper, P. M. Shoolingin-Jordan, and R. Neier. 2001. The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors. FEBS Lett. 503:196–200.[PubMed] [CrossRef]
70. Erskine, P. T., E. Norton, J. B. Cooper, R. Lambert, A. Coker, G. Lewis, P. Spencer, M. Sarwar, S. P. Wood, M. J. Warren, and P. M. Shoolingin-Jordan. 1999. X-ray structure of 5-aminolevulinic acid dehydratase from Escherichia coli complexed with the inhibitor levulinic acid at 2.0 Å resolution. Biochemistry 38:4266–4276.[PubMed] [CrossRef]
71. Escalante-Semerena, J. C. 1999. Regulation of adenosylcobalamin biosynthesis in Salmonella typhimurium, p. 577–594. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York, NY.
72. Escalante-Semerena, J. C., and J. R. Roth. 1987. Regulation of cobalamin biosynthetic operons in Salmonella typhimurium. J. Bacteriol. 169:2251–2258.[PubMed]
73. Escalante-Semerena, J. C., S. J. Suh, and J. R. Roth. 1990. cobA function is required for both de novo cobalamin biosynthesis and assimilation of exogenous corrinoids in Salmonella typhimurium. J. Bacteriol. 172:273–280.[PubMed]
74. Evans, J. C., D. P. Huddler, M. T. Hilgers, G. Romanchuk, R. G. Matthews, and M. L. Ludwig. 2004. Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase. Proc. Natl. Acad. Sci. USA 101:3729–3736.[PubMed] [CrossRef]
75. Faust, L. R., J. A. Connor, D. M. Roof, J. A. Hoch, and B. M. Babior. 1990. Cloning, sequencing, and expression of the genes encoding the adenosylcobalamin-dependent ethanolamine ammonia-lyase of Salmonella typhimurium. J. Biol. Chem. 265:12462–12466.[PubMed]
76. Fazzio, T. G., and J. R. Roth. 1996. Evidence that the CysG protein catalyzes the first reaction specific to B12 synthesis in Salmonella typhimurium, insertion of cobalt. J. Bacteriol. 178:6952–6959.[PubMed]
77. Fonseca, M. V., N. R. Buan, A. R. Horswill, I. Rayment, and J. C. Escalante-Semerena. 2002. The ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica requires the 2'-OH group of ATP for function and yields inorganic triphosphate as its reaction byproduct. J. Biol. Chem. 277:33127–33131.[PubMed] [CrossRef]
78. Fonseca, M. V., and J. C. Escalante-Semerena. 2001. An in vitro reducing system for the enzymic conversion of cobalamin to adenosylcobalamin. J. Biol. Chem. 276:32101–32108.[PubMed] [CrossRef]
79. Fonseca, M. V., and J. C. Escalante-Semerena. 2000. Reduction of cob(III)alamin to cob(II)alamin in Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 182:4304–4309.[PubMed] [CrossRef]
80. Ford, J. E., E. S. Holdsworth, and S. K. Kon. 1955. The biosynthesis of vitamin B12-like compounds. Biochem. J. 59:86–93.[PubMed]
81. Ford, J. E., and S. H. Hutner. 1955. Role of vitamin B12 in the metabolism of microorganisms. Vitam. Horm. 1955:101–136. [CrossRef]
82. Frank, S., E. Deery, A. A. Brindley, H. K. Leech, A. Lawrence, P. Heathcote, H. L. Schubert, K. Brocklehurst, S. E. Rigby, M. J. Warren, and R. W. Pickersgill. 2007. Elucidation of substrate specificity in the cobalamin (vitamin B12) biosynthetic methyltransferases. Structure and function of the C20 methyltransferase (CbiL) from Methanothermobacter thermautotrophicus. J. Biol. Chem. 282:23957–23969.[PubMed] [CrossRef]
83. Fraústo da Silva, J. J. R., and R. J. P. Williams. 2001. The Biological Chemistry of the Elements, 2nd ed. Oxford University Press, Oxford, United Kingdom.
84. Fresquet, V., L. Williams, and F. M. Raushel. 2004. Mechanism of cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium LT2. Biochemistry 43:10619–10627.[PubMed] [CrossRef]
85. Reference deleted.
86. Friedmann, H. C. 1965. Partial purification and properties of a single displacement trans-N-glycosidase. J. Biol. Chem. 240:413–418.[PubMed]
87. Friedmann, H. C. 1968. Vitamin B12 biosynthesis. Evidence for a new precursor vitamin B12 5'-phosphate. J. Biol. Chem. 243:2065–2075.[PubMed]
88. Friedmann, H. C., and D. L. Harris. 1965. The formation of α-glycosidic 5'-nucleotides by a single displacement trans-N-glycosidase. J. Biol. Chem. 240:406–412.[PubMed]
89. Friedmann, H. C., and R. K. Thauer (ed.). 1992. Macrocyclic Tetrapyrrole Biosynthesis in Bacteria, vol. 3. Academic Press, Inc., New York, NY.
90. Gantt, E., and S. F. Conti. 1969. Ultrastructure of blue-green algae. J. Bacteriol. 97:1486–1493.[PubMed]
91. Gerfen, G. J. 1999. EPR spectroscopy of B12-dependent enzymes, p. 165–195. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc, New York, NY.
92. Glusker, J. P. 1982. X-ray crystallography of B12 and cobaloximes, p. 23–106. In D. Dolphin (ed.), B12, vol. 1. John Wiley & Sons, New York, NY.
93. Gonzalez, J. C., K. Peariso, J. E. Penner-Hahn, and R. G. Matthews. 1996. Cobalamin-independent methionine synthase from Escherichia coli: a zinc metalloenzyme. Biochemistry 35:12228–12234.[PubMed] [CrossRef]
94. Grabau, C., and J. R. Roth. 1992. A Salmonella typhimurium cobalamin-deficient mutant blocked in 1-amino-2-propanol synthesis. J. Bacteriol. 174:2138–2144.[PubMed]
95. Gray, M. J., and J. C. Escalante-Semerena. 2007. Single-enzyme conversion of FMNH2 to 5,6-dimethylbenzimidazole, the lower ligand of B12. Proc. Natl. Acad. Sci. USA 104:2921–2926.[PubMed] [CrossRef]
96. Greene, R. C. 1996. Biosynthesis of methionine, p. 542–560. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed. ASM Press, Washington, DC.
97. Hall, D. A., T. C. Jordan-Starck, R. O. Loo, M. L. Ludwig, and R. G. Matthews. 2000. Interaction of flavodoxin with cobalamin-dependent methionine synthase. Biochemistry 39:10711–10719.[PubMed] [CrossRef]
98. Haller, T., T. Buckel, J. Rétey, and J. A. Gerlt. 2000. Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli. Biochemistry 39:4622–4629.[PubMed] [CrossRef]
99. Hart, G. J., A. D. Miller, F. J. Leeper, and A. R. Battersby. 1987. Biosynthesis of the natural porphyrins: proof that hydroxymethylbilane synthase (prophobilinogen deaminase) uses a novel binding group in its catalytic action. J. Chem. Soc. Chem. Commun. 23:1762–1764. [CrossRef]
100. Havemann, G. D., and T. A. Bobik. 2003. Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 185:5086–5095.[PubMed] [CrossRef]
101. Havemann, G. D., E. M. Sampson, and T. A. Bobik. 2002. PduA is a shell protein of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 184:1253–1261.[PubMed] [CrossRef]
102. Hennig, M., B. Grimm, R. Contestabile, R. A. John, and J. N. Jansonius. 1997. Crystal structure of glutamate-1-semialdehyde aminomutase: an α2-dimeric vitamin B6–dependent enzyme with asymmetry in structure and active site reactivity. Proc. Natl. Acad. Sci. USA 94:4866–4871.[PubMed] [CrossRef]
103. Hodgkin, D. C. 1979. New and old problems in the structure analysis of vitamin B12, p. 19–36. In B. Zagalak and W. Friedrich (ed.), Vitamin B12. de Gruyter, Berlin, Germany.
104. Hondorp, E. R., and R. G. Matthews. 2004. Oxidative stress inactivates cobalamin-independent methionine synthase (MetE) in Escherichia coli. PLoS Biol. 2:e336. [CrossRef]
105. Hoover, D. M., J. T. Jarrett, R. H. Sands, W. R. Dunham, M. L. Ludwig, and R. G. Matthews. 1997. Interaction of Escherichia coli cobalamin-dependent methionine synthase and its physiological partner flavodoxin: binding of flavodoxin leads to axial ligand dissociation from the cobalamin cofactor. Biochemistry 36:127–138.[PubMed] [CrossRef]
106. Hoover, D. M., and M. L. Ludwig. 1997. A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 Å resolution. Protein Sci. 6:2525–2537.[PubMed] [CrossRef]
107. Horswill, A. R., and J. C. Escalante-Semerena. 2001. In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate. Biochemistry 40:4703–4713.[PubMed] [CrossRef]
108. Horswill, A. R., and J. C. Escalante-Semerena. 1997. Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon. J. Bacteriol. 179:928–940.[PubMed]
109. Horswill, A. R., and J. C. Escalante-Semerena. 1999. Salmonella typhimurium LT2 catabolizes propionate via the 2-methylcitric acid cycle. J. Bacteriol. 181:5615–5623.[PubMed]
110. Huennekens, F. M., K. S. Vitols, K. Fujii, and D. W. Jacobsen. 1982. Biosynthesis of cobalamin coenzymes, p. 145–168. In D. Dolphin (ed.), B12, vol. 1. John Wiley & Sons, Inc., New York, NY.
111. Ilag, L. L., and D. Jahn. 1992. Activity and spectroscopic properties of the Escherichia coli glutamate 1-semialdehyde aminotransferase and the putative active site mutant K265R. Biochemistry 31:7143–7151.[PubMed] [CrossRef]
112. Ilag, L. L., D. Jahn, G. Eggertsson, and D. Soll. 1991. The Escherichia coli hemL gene encodes glutamate 1-semialdehyde aminotransferase. J. Bacteriol. 173:3408–3413.[PubMed]
113. Imai, S., C. M. Armstrong, M. Kaeberlein, and L. Guarente. 2000. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403:795–800.[PubMed] [CrossRef]
114. International Union of Pure and Applied Chemistry-International Union of Biochemistry and Molecular Biology Joint Commission on Biochemical Nomenclature. 1966. Tentative rules. Nomenclature of corrinoids. Biochim. Biophys. Acta 117:285–288.[PubMed]
115. International Union of Pure and Applied Chemistry-International Union of Biochemistry and Molecular Biology Joint Commission on Biochemical Nomenclature. 1974. The nomenclature of corrinoids (1973 recommendations). Biochemistry 13:1555–1560.[PubMed] [CrossRef]
116. Irion, E., and L. G. Ljungdahl. 1965. Isolation of factor III coenzyme and cobyric acid coenzyme plus other B12 factors from Clostridium thermoaceticum. Biochemistry 4:2780–2790.[PubMed] [CrossRef]
117. Iuchi, S., and E. C. Lin. 1993. Adaptation of Escherichia coli to redox environments by gene expression. Mol. Microbiol. 9:9–15.[PubMed] [CrossRef]
118. Jahn, D., U. Michelsen, and D. Soll. 1991. Two glutamyl-tRNA reductase activities in Escherichia coli. J. Biol. Chem. 266:2542–2548.[PubMed]
119. Jahn, D., E. Verkamp, and D. Soll. 1992. Glutamyl-transfer RNA: a precursor of heme and chlorophyll biosynthesis. Trends Biochem. Sci. 17:215–218.[PubMed] [CrossRef]
120. Jarrett, J. T., D. M. Hoover, M. L. Ludwig, and R. G. Matthews. 1998. The mechanism of adenosylmethionine-dependent activation of methionine synthase: a rapid kinetic analysis of intermediates in reductive methylation of cob(II)alamin enzyme. Biochemistry 37:12649–12658.[PubMed] [CrossRef]
121. Jeter, R. M. 1990. Cobalamin-dependent 1,2-propanediol utilization by Salmonella typhimurium. J. Gen. Microbiol. 136:887–896.[PubMed]
122. Jeter, R. M., B. M. Olivera, and J. R. Roth. 1984. Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions. J. Bacteriol. 159:206–213.[PubMed]
123. Jeter, R. M., and J. R. Roth. 1987. Cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium. J. Bacteriol. 169:3189–3198.[PubMed]
124. Johnson, C. L., M. L. Buszko, and T. A. Bobik. 2004. Purification and initial characterization of the Salmonella enterica PduO ATP:cob(I)alamin adenosyltransferase. J. Bacteriol. 186:7881–7887.[PubMed] [CrossRef]
125. Johnson, C. L., E. Pechonick, S. D. Park, G. D. Havemann, N. A. Leal, and T. A. Bobik. 2001. Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene. J. Bacteriol. 183:1577–1584.[PubMed] [CrossRef]
126. Johnson, M. G., and J. C. Escalante-Semerena. 1992. Identification of 5,6-dimethylbenzimidazole as the Co α ligand of the cobamide synthesized by Salmonella typhimurium. Nutritional characterization of mutants defective in biosynthesis of the imidazole ring. J. Biol. Chem. 267:13302–13305.[PubMed]
127. Jordan, P. M., and J. S. Seehra. 1980. 13C NMR as a probe for the study of enzyme-catalysed reactions: mechanism of action of 5-aminolevulinic acid dehydratase. FEBS Lett. 114:283–286.[PubMed] [CrossRef]
128. Jordan, P. M., and J. S. Seehra. 1979. The biosynthesis of uroporphyrinogen III: order of assembly of the four porphobilinogen molecules in the formation of the tetrapyrrole ring. FEBS Lett. 104:364–366.[PubMed] [CrossRef]
129. Jordan, P. M., and M. J. Warren. 1987. Evidence for a dipyrromethane cofactor at the catalytic site of E. coli porphobilinogen deaminase. FEBS Lett. 225:87–92.[PubMed] [CrossRef]
130. Kajiwara, Y., P. J. Santander, C. A. Roessner, L. M. Perez, and A. I. Scott. 2006. Genetically engineered synthesis and structural characterization of cobalt-precorrin 5A and -5B, two new intermediates on the anaerobic pathway to vitamin B12: definition of the roles of the CbiF and CbiG enzymes. J. Am. Chem. Soc. 128:9971–9978.[PubMed] [CrossRef]
131. Kaneko, Y., R. Danev, K. Nagayama, and H. Nakamoto. 2006. Intact carboxysomes in a cyanobacterial cell visualized by Hilbert differential contrast transmission electron microscopy. J. Bacteriol. 188:805–808.[PubMed] [CrossRef]
132. Kannangara, C. G., S. P. Gough, P. Bruyant, J. K. Hoober, A. Kahn, and D. von Wettstein. 1988. tRNA(Glu) as a cofactor in delta-aminolevulinate biosynthesis: steps that regulate chlorophyll synthesis. Trends Biochem. Sci. 13:139–143.[PubMed] [CrossRef]
133. Keck, B., M. Munder, and P. Renz. 1998. Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety. Arch. Microbiol. 171:66–68.[PubMed] [CrossRef]
134. Keck, B., and P. Renz. 2000. Salmonella typhimurium forms adenylcobamide and 2-methyladenylcobamide, but no detectable cobalamin during strictly anaerobic growth. Arch. Microbiol. 173:76–77.[PubMed] [CrossRef]
135. Keller, J. P., P. M. Smith, J. Benach, D. Christendat, G. T. deTitta, and J. F. Hunt. 2002. The crystal structure of MT0146/CbiT suggests that the putative precorrin-8w decarboxylase is a methyltransferase. Structure 10:1475–1487.[PubMed] [CrossRef]
136. Kerfeld, C. A., M. R. Sawaya, S. Tanaka, C. V. Nguyen, M. Phillips, M. Beeby, and T. O. Yeates. 2005. Protein structures forming the shell of primitive bacterial organelles. Science 309:936–938.[PubMed] [CrossRef]
137. Kim, W., T. A. Major, and W. B. Whitman. 2005. Role of the precorrin 6-X reductase gene in cobamide biosynthesis in Methanococcus maripaludis. Archaea 1:375–384.[PubMed] [CrossRef]
138. Kofoid, E., C. Rappleye, I. Stojiljkovic, and J. Roth. 1999. The 17-gene ethanolamine (eut) operon of Salmonella typhimurium encodes five homologues of carboxysome shell proteins. J. Bacteriol. 181:5317–5329.[PubMed]
139. Krathy, C., and B. Kräutler. 1999. X-ray crystallography of B12, p. 9–41. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York, NY.
140. Kräutler, B., H. P. Kohler, and E. Stupperich. 1988. 5'-Methylbenzimidazolyl-cobamides are the corrinoids from some sulfate-reducing and sulfur-metabolizing bacteria. Eur. J. Biochem. 176:461–469.[PubMed] [CrossRef]
141. Kräutler, B., J. Moll, and R. K. Thauer. 1987. The corrinoid from Methanobacterium thermoautotrophicum (Marburg strain). Spectroscopic structure analysis and identification as Co beta-cyano-5'-hydroxybenzimidazolyl-cobamide (factor III). Eur. J. Biochem. 162:275–278.[PubMed] [CrossRef]
142. Kulkarni, P. R., X. Cui, J. W. Williams, A. M. Stevens, and R. V. Kulkarni. 2006. Prediction of CsrA-regulating small RNAs in bacteria and their experimental verification in Vibrio fischeri. Nucleic Acids Res. 34:3361–3369.[PubMed] [CrossRef]
143. Lawhon, S. D., J. G. Frye, M. Suyemoto, S. Porwollik, M. McClelland, and C. Altier. 2003. Global regulation by CsrA in Salmonella typhimurium. Mol. Microbiol. 48:1633–1645.[PubMed] [CrossRef]
144. Lawrence, J. G., and J. R. Roth. 1996. Evolution of coenzyme B12 synthesis among enteric bacteria: evidence for loss and reacquisition of a multigene complex. Genetics 142:11–24.[PubMed]
145. Lawrence, J. G., and J. R. Roth. 1995. The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli. J. Bacteriol. 177:6371–6380.[PubMed]
146. Leal, N. A., G. D. Havemann, and T. A. Bobik. 2003. PduP is a coenzyme-A-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2. Arch. Microbiol. 180:353–361.[PubMed] [CrossRef]
147. Leal, N. A., H. Olteanu, R. Banerjee, and T. A. Bobik. 2004. Human ATP:cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase. J. Biol. Chem. 279:47536–47542.[PubMed] [CrossRef]
148. Leal, N. A., S. D. Park, P. E. Kima, and T. A. Bobik. 2003. Identification of the human and bovine ATP:cob(I)alamin adenosyltransferase cDNAs based on complementation of a bacterial mutant. J. Biol. Chem. 278:9227–9234.[PubMed] [CrossRef]
149. Leech, H. K., E. Raux, K. J. McLean, A. W. Munro, N. J. Robinson, G. P. Borrelly, M. Malten, D. Jahn, S. E. Rigby, P. Heathcote, and M. J. Warren. 2003. Characterization of the cobaltochelatase CbiXL: evidence for a 4Fe-4S center housed within an MXCXXC motif. J. Biol. Chem. 278:41900–4197.[PubMed] [CrossRef]
150. Leipe, D. D., Y. I. Wolf, E. V. Koonin, and L. Aravind. 2002. Classification and evolution of P-loop GTPases and related ATPases. J. Mol. Biol. 317:41–72.[PubMed] [CrossRef]
151. Lexa, D., and J.-M. Saveant. 1976. Electrochemistry of vitamin B12. 1. Role of the base-on/base-off reaction in the oxidoreduction mechanism of the B12r-B12s system. J. Am. Chem. Soc. 98:2652–2658.[PubMed] [CrossRef]
152. Lexa, D., J. M. Saveant, and J. Zickler. 1977. Electrochemistry of vitamin B12. 2. Redox and acid-base equilibria in the B12a/B12r system. J. Am. Chem. Soc. 99:2786–2790.[PubMed] [CrossRef]
153. Li, J. M., O. Brathwaite, S. D. Cosloy, and C. S. Russell. 1989. 5-Aminolevulinic acid synthesis in Escherichia coli. J. Bacteriol. 171:2547–2552.[PubMed]
154. Li, J. M., C. S. Russell, and S. D. Cosloy. 1989. The structure of the Escherichia coli hemB gene. Gene 75:177–184.[PubMed] [CrossRef]
155. Liu, Y., N. A. Leal, E. M. Sampson, C. L. Johnson, G. D. Havemann, and T. A. Bobik. 2007. PduL is an evolutionarily distinct phosphotransacylase involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 189:1589–1596.[PubMed] [CrossRef]
156. Louie, G. V., P. D. Brownlie, R. Lambert, J. B. Cooper, T. L. Blundell, S. P. Wood, M. J. Warren, S. C. Woodcock, and P. M. Jordan. 1992. Structure of porphobilinogen deaminase reveals a flexible multidomain polymerase with a single catalytic site. Nature 359:33–39.[PubMed] [CrossRef]
157. Ludwig, M. L., and R. G. Matthews. 1997. Structure-based perspectives on B12-dependent enzymes. Annu. Rev. Biochem. 66:269–313.[PubMed] [CrossRef]
158. Luer, C., S. Schauer, K. Mobius, J. Schulze, W. D. Schubert, D. W. Heinz, D. Jahn, and J. Moser. 2005. Complex formation between glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase in Escherichia coli during the initial reactions of porphyrin biosynthesis. J. Biol. Chem. 280:18568–18572.[PubMed] [CrossRef]
159. Lundrigan, M. D., L. C. De Veaux, B. J. Mann, and R. J. Kadner. 1987. Separate regulatory systems for the repression of metE and btuB by vitamin B12 in Escherichia coli. Mol. Gen. Genet. 206:401–407.[PubMed] [CrossRef]
160. Lundrigan, M. D., and R. J. Kadner. 1989. Altered cobalamin metabolism in Escherichia coli btuR mutants affects btuB gene regulation. J. Bacteriol. 171:154–161.[PubMed]
161. Lundrigan, M. D., W. Koster, and R. J. Kadner. 1991. Transcribed sequences of the Escherichia coli btuB gene control its expression and regulation by vitamin B12. Proc. Natl. Acad. Sci. USA 88:1479–1483.[PubMed] [CrossRef]
162. Luschinsky, C. L., J. T. Drummond, R. G. Matthews, and M. L. Ludwig. 1992. Crystallization and preliminary X-ray diffraction studies of the cobalamin-binding domain of methionine synthase from Escherichia coli. J. Mol. Biol. 225:557–560.[PubMed] [CrossRef]
163. Maggio-Hall, L. A., K. R. Claas, and J. C. Escalante-Semerena. 2004. The last step in coenzyme B12 synthesis is localized to the cell membrane in bacteria and archaea. Microbiology 150:1385–1395.[PubMed] [CrossRef]
164. Maggio-Hall, L. A., P. C. Dorrestein, J. C. Escalante-Semerena, and T. P. Begley. 2003. Formation of the dimethylbenzimidazole ligand of coenzyme B12 under physiological conditions by a facile oxidative cascade. Org. Lett. 5:2211–2213.[PubMed] [CrossRef]
165. Maggio-Hall, L. A., and J. C. Escalante-Semerena. 2003. α-5,6-Dimethylbenzimidazole adenine dinucleotide (α-DAD), a putative new intermediate of coenzyme B12 biosynthesis in Salmonella typhimurium. Microbiology 149:983–990.[PubMed] [CrossRef]
166. Maggio-Hall, L. A., and J. C. Escalante-Semerena. 1999. In vitro synthesis of the nucleotide loop of cobalamin by Salmonella typhimurium enzymes. Proc. Natl. Acad. Sci. USA 96:11798–11803.[PubMed] [CrossRef]
167. Malpica, R., G. R. Sandoval, C. Rodriguez, B. Franco, and D. Georgellis. 2006. Signaling by the arc two-component system provides a link between the redox state of the quinone pool and gene expression. Antioxid. Redox Signal. 8:781–795.[PubMed] [CrossRef]
168. Marques, H. M. 1999. Modeling the structure of cobalt corrins by molecular dynamics methods, p. 289–313. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York, NY.
169. Mathews, M. A., H. L. Schubert, F. G. Whitby, K. J. Alexander, K. Schadick, H. A. Bergonia, J. D. Phillips, and C. P. Hill. 2001. Crystal structure of human uroporphyrinogen III synthase. EMBO J. 20:5832–5839.[PubMed] [CrossRef]
170. Matthews, R. G. 2001. Cobalamin-dependent methyltransferases. Acc. Chem. Res. 34:681–689.[PubMed] [CrossRef]
171. Mazumder, T. K., N. Nishio, M. Hayashi, and S. Nagai. 1986. Production of corrinoids by Methanosarcina barkeri growing on methanol. Biotechnol. Lett. 8:843–848. [CrossRef]
172. Mercante, J., K. Suzuki, X. Cheng, P. Babitzke, and T. Romeo. 2006. Comprehensive alanine-scanning mutagenesis of Escherichia coli CsrA defines two subdomains of critical functional importance. J. Biol. Chem. 281:31832–31842.[PubMed] [CrossRef]
173. Mitchell, L. W., and E. K. Jaffe. 1993. Porphobilinogen synthase from Escherichia coli is a Zn(II) metalloenzyme stimulated by Mg(II). Arch. Biochem. Biophys. 300:169–177.[PubMed] [CrossRef]
174. Model, P., G. Jovanovic, and J. Dworkin. 1997. The Escherichia coli phage-shock-protein (psp) operon. Mol. Microbiol. 24:255–261.[PubMed] [CrossRef]
175. Mori, K., R. Bando, N. Hieda, and T. Toraya. 2004. Identification of a reactivating factor for adenosylcobalamin-dependent ethanolamine ammonia lyase. J. Bacteriol. 186:6845–6854.[PubMed] [CrossRef]
176. Moser, J., W. D. Schubert, V. Beier, I. Bringemeier, D. Jahn, and D. W. Heinz. 2001. V-shaped structure of glutamyl-tRNA reductase, the first enzyme of tRNA-dependent tetrapyrrole biosynthesis. EMBO J. 20:6583–6590.[PubMed] [CrossRef]
177. Nahvi, A., J. E. Barrick, and R. R. Breaker. 2004. Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res. 32:143–150.[PubMed] [CrossRef]
178. Nahvi, A., N. Sudarsan, M. S. Ebert, X. Zou, K. L. Brown, and R. R. Breaker. 2002. Genetic control by a metabolite binding mRNA. Chem. Biol. 9:1043–1049.[PubMed] [CrossRef]
179. Nou, X., and R. J. Kadner. 2000. Adenosylcobalamin inhibits ribosome binding to btuB RNA. Proc. Natl. Acad. Sci. USA 97:7190–7195.[PubMed] [CrossRef]
180. Orus, M. I., M. L. Rodriguez, F. Martinez, and E. Marco. 1995. Biogenesis and ultrastructure of carboxysomes from wild type and mutants of Synechococcus sp. strain PCC 7942. Plant Physiol. 107:1159–1166.[PubMed]
181. O’Toole, G. A. 1994. Biochemistry and genetics of cobalamin nucleotide loop assembly in Salmonella typhimurium. Ph.D. dissertation. University of Wisconsin, Madison, WI.
182. O’Toole, G. A., and J. C. Escalante-Semerena. 1993. cobU-dependent assimilation of nonadenosylated cobinamide in cobA mutants of Salmonella typhimurium. J. Bacteriol. 175:6328–6336.[PubMed]
183. O’Toole, G. A., and J. C. Escalante-Semerena. 1995. Purification and characterization of the bifunctional CobU enzyme of Salmonella typhimurium LT2. Evidence for a CobU-GMP intermediate. J. Biol. Chem. 270:23560–23569.[PubMed] [CrossRef]
184. O’Toole, G. A., M. R. Rondon, and J. C. Escalante-Semerena. 1993. Analysis of mutants of Salmonella typhimurium defective in the synthesis of the nucleotide loop of cobalamin. J. Bacteriol. 175:3317–3326.[PubMed]
185. O’Toole, G. A., J. R. Trzebiatowski, and J. C. Escalante-Semerena. 1994. The cobC gene of Salmonella typhimurium codes for a novel phosphatase involved in the assembly of the nucleotide loop of cobalamin. J. Biol. Chem. 269:26503–26511.[PubMed]
186. Palacios, S., V. J. Starai, and J. C. Escalante-Semerena. 2003. Propionyl coenzyme A is a common intermediate in the 1,2-propanediol and propionate catabolic pathways needed for expression of the prpBCDE operon during growth of Salmonella enterica on 1,2-propanediol. J. Bacteriol. 185:2802–2810.[PubMed] [CrossRef]
187. Peakman, T., J. Crouzet, J. F. Mayaux, S. Busby, S. Mohan, N. Harborne, J. Wootton, R. Nicolson, and J. Cole. 1990. Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-12 chromosome. Eur. J. Biochem. 191:315–323.[PubMed] [CrossRef]
188. Peariso, K., Z. S. Zhou, A. E. Smith, R. G. Matthews, and J. E. Penner-Hahn. 2001. Characterization of the zinc sites in cobalamin-independent and cobalamin-dependent methionine synthase using zinc and selenium X-ray absorption spectroscopy. Biochemistry 40:987–993.[PubMed] [CrossRef]
189. Pejchal, R., and M. L. Ludwig. 2005. Cobalamin-independent methionine synthase (MetE): a face-to-face double barrel that evolved by gene duplication. PLoS Biol. 3:e31. [CrossRef]
190. Penrod, J. T., C. C. Mace, and J. R. Roth. 2004. A pH-sensitive function and phenotype: evidence that EutH facilitates diffusion of uncharged ethanolamine in Salmonella enterica. J. Bacteriol. 186:6885–6890.[PubMed] [CrossRef]
191. Plano, G. V. 2004. Modulation of AraC family member activity by protein ligands. Mol. Microbiol. 54:287–290.[PubMed] [CrossRef]
192. Pollich, M., C. Wersig, and G. Klug. 1996. The bluF gene of Rhodobacter capsulatus is involved in conversion of cobinamide to cobalamin (vitamin B12). J. Bacteriol. 178:7308–7310.[PubMed]
193. Price-Carter, M., J. Tingey, T. A. Bobik, and J. R. Roth. 2001. The alternative electron acceptor tetrathionate supports B12-dependent anaerobic growth of Salmonella enterica serovar Typhimurium on ethanolamine or 1,2-propanediol. J. Bacteriol. 183:2463–2475.[PubMed] [CrossRef]
194. Raux, E., A. Lanois, F. Levillayer, M. J. Warren, E. Brody, A. Rambach, and C. Thermes. 1996. Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: functional studies in S. typhimurium and Escherichia coli. J. Bacteriol. 178:753–767.[PubMed]
195. Raux, E., H. K. Leech, R. Beck, H. L. Schubert, P. J. Santander, C. A. Roessner, A. I. Scott, J. H. Martens, D. Jahn, C. Thermes, A. Rambach, and M. J. Warren. 2003. Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium. Biochem. J. 370:505–516.[PubMed] [CrossRef]
196. Raux, E., T. McVeigh, S. E. Peters, T. Leustek, and M. J. Warren. 1999. The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis. Biochem. J. 338:701–708.[PubMed] [CrossRef]
197. Raux, E., H. L. Schubert, J. M. Roper, K. S. Wilson, and M. J. Warren. 1999. Vitamin B-12: insights into biosynthesis’s mount improbable. Bioorg. Chem. 27:100–118. [CrossRef]
198. Raux, E., H. L. Schubert, and M. J. Warren. 2000. Biosynthesis of cobalamin (vitamin B12): a bacterial conundrum. Cell. Mol. Life Sci. 57:1880–1893.[PubMed] [CrossRef]
199. Raux, E., C. Thermes, P. Heathcote, A. Rambach, and M. J. Warren. 1997. A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis. J. Bacteriol. 179:3202–3212.[PubMed]
200. Raux, E. 1998. Ph.D. thesis. University of London, London, United Kingdom.
201. Ravnum, S., and D. I. Andersson. 2001. An adenosyl-cobalamin (coenzyme-B12)-repressed translational enhancer in the cob mRNA of Salmonella typhimurium. Mol. Microbiol. 39:1585–1594.[PubMed] [CrossRef]
202. Ravnum, S., and D. I. Andersson. 1997. Vitamin B12 repression of the btuB gene in Salmonella typhimurium is mediated via a translational control which requires leader and coding sequences. Mol. Microbiol. 23:35–42.[PubMed] [CrossRef]
203. Renz, P. 1999. Biosynthesis of the 5,6-dimethylbenzimidazole moiety of cobalamin and of other bases found in natural corrinoids, p. 557–575. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York, NY.
204. Richter-Dahlfors, A. A., and D. I. Andersson. 1991. Analysis of an anaerobically induced promoter for the cobalamin biosynthetic genes in Salmonella typhimurium. Mol. Microbiol. 5:1337–1345.[PubMed] [CrossRef]
205. Richter-Dahlfors, A. A., and D. I. Andersson. 1992. Cobalamin (vitamin B12) repression of the Cob operon in Salmonella typhimurium requires sequences within the leader and the first translated open reading frame. Mol. Microbiol. 6:743–749.[PubMed] [CrossRef]
206. Rife, C., R. Schwarzenbacher, D. McMullan, P. Abdubek, E. Ambing, H. Axelrod, T. Biorac, J. M. Canaves, H. J. Chiu, A. M. Deacon, M. DiDonato, M. A. Elsliger, A. Godzik, C. Grittini, S. K. Grzechnik, J. Hale, E. Hampton, G. W. Han, J. Haugen, M. Hornsby, L. Jaroszewski, H. E. Klock, E. Koesema, A. Kreusch, P. Kuhn, S. A. Lesley, M. D. Miller, K. Moy, E. Nigoghossian, J. Paulsen, K. Quijano, R. Reyes, E. Sims, G. Spraggon, R. C. Stevens, H. van den Bedem, J. Velasquez, J. Vincent, A. White, G. Wolf, Q. Xu, K. O. Hodgson, J. Wooley, and I. A. Wilson. 2005. Crystal structure of the global regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a new fold. Proteins 61:449–453.[PubMed] [CrossRef]
207. Rodionov, D. A., A. G. Vitreschak, A. A. Mironov, and M. S. Gelfand. 2003. Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes. J. Biol. Chem. 278:41148–41159.[PubMed] [CrossRef]
208. Roessner, C. A., and A. I. Scott. 2006. Fine-tuning our knowledge of the anaerobic route to cobalamin (vitamin B12). J. Bacteriol. 188:7331–7334.[PubMed] [CrossRef]
209. Roessner, C. A., M. J. Warren, P. J. Santander, B. P. Atshaves, S. Ozaki, N. J. Stolowich, K. Iida, and A. I. Scott. 1992. Expression of 9 Salmonella typhimurium enzymes for cobinamide synthesis. Identification of the 11-methyl and 20-methyl transferases of corrin biosynthesis. FEBS Lett. 301:73–78.[PubMed] [CrossRef]
210. Roessner, C. A., H. J. Williams, and A. I. Scott. 2005. Genetically engineered production of 1-desmethylcobyrinic acid, 1-desmethylcobyrinic acid a,c-diamide, and cobyrinic acid a,c-diamide in E. coli implies a role for CbiD in C-1 methylation in the anaerobic pathway to cobalamin. J. Biol. Chem. 280:16748–16753.[PubMed] [CrossRef]
211. Roessner, C. A., H. J. Williams, and A. I. Scott. 2005. Genetically engineered production of 1-desmethylcobyrinic acid, 1-desmethylcobyrinic acid a,c-diamide, and cobyrinic acid a,c-diamide in Escherichia coli implies a role for CbiD in C-1 methylation in the anaerobic pathway to cobalamin. J. Biol. Chem. 280:16748–16753.[PubMed] [CrossRef]
212. Romeo, T. 1998. Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB. Mol. Microbiol. 29:1321–1330.[PubMed] [CrossRef]
213. Romeo, T. 1996. Post-transcriptional regulation of bacterial carbohydrate metabolism: evidence that the gene product CsrA is a global mRNA decay factor. Res. Microbiol. 147:505–512.[PubMed] [CrossRef]
214. Romeo, T., M. Gong, M. Y. Liu, and A. M. Brun-Zinkernagel. 1993. Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties. J. Bacteriol. 175:4744–4755.[PubMed]
215. Rondon, M. R., and J. C. Escalante-Semerena. 1998. High levels of transcription factor RpoS (sigma S) in mviA mutants negatively affect 1,2-propanediol-dependent transcription of the cob/pdu regulon of Salmonella typhimurium LT2. FEMS Microbiol. Lett. 169:147–153.[PubMed] [CrossRef]
216. Rondon, M. R., and J. C. Escalante-Semerena. 1996. In vitro analysis of the interactions between the PocR regulatory protein and the promoter region of the cobalamin biosynthetic (cob) operon of Salmonella typhimurium LT2. J. Bacteriol. 178:2196–2203.[PubMed]
217. Rondon, M. R., and J. C. Escalante-Semerena. 1997. Integration host factor is required for 1,2-propanediol-dependent transcription of the cob/pdu regulon in Salmonella typhimurium LT2. J. Bacteriol. 179:3797–3800.[PubMed]
218. Rondon, M. R., and J. C. Escalante-Semerena. 1992. The poc locus is required for 1,2-propanediol-dependent transcription of the cobalamin biosynthetic (cob) and propanediol utilization (pdu) genes of Salmonella typhimurium. J. Bacteriol. 174:2267–2272.[PubMed]
219. Rondon, M. R., A. R. Horswill, and J. C. Escalante-Semerena. 1995. DNA polymerase I function is required for the utilization of ethanolamine, 1,2-propanediol, and propionate by Salmonella typhimurium LT2. J. Bacteriol. 177:7119–7124.[PubMed]
220. Rondon, M. R., R. Kazmierczak, and J. C. Escalante-Semerena. 1995. Glutathione is required for maximal transcription of the cobalamin biosynthetic and 1,2-propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, 1,2-propanediol, and propionate in Salmonella typhimurium LT2. J. Bacteriol. 177:5434–5439.[PubMed]
221. Ronzio, R. A., and H. A. Barker. 1967. Enzymic synthesis of guanosine diphosphate cobinamide by extracts of propionic acid bacteria. Biochemistry 6:2344–2354.[PubMed] [CrossRef]
222. Roof, D. M., and J. R. Roth. 1992. Autogenous regulation of ethanolamine utilization by a transcriptional activator of the eut operon in Salmonella typhimurium. J. Bacteriol. 174:6634–6643.[PubMed]
223. Roof, D. M., and J. R. Roth. 1988. Ethanolamine utilization in Salmonella typhimurium. J. Bacteriol. 170:3855–3863.[PubMed]
224. Roof, D. M., and J. R. Roth. 1989. Functions required for vitamin B12-dependent ethanolamine utilization in Salmonella typhimurium. J. Bacteriol. 171:3316–3323.[PubMed]
225. Roth, J. R., J. G. Lawrence, and T. A. Bobik. 1996. Cobalamin (coenzyme B12): synthesis and biological significance. Annu. Rev. Microbiol. 50:137–181.[PubMed] [CrossRef]
226. Roth, J. R., J. G. Lawrence, M. Rubenfield, S. Kieffer-Higgins, and G. M. Church. 1993. Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium. J. Bacteriol. 175:3303–3316.[PubMed]
227. Roy, I., and P. F. Leadlay. 1992. Physical map location of the new Escherichia coli gene sbm. J. Bacteriol. 174:5763–5764.[PubMed]
228. Sampson, E. M., C. L. Johnson, and T. A. Bobik. 2005. Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase. Microbiology 151:1169–1177.[PubMed] [CrossRef]
229. Sanishvili, R., M. Pennycooke, J. Gu, X. Xu, A. Joachimiak, A. M. Edwards, and D. Christendat. 2004. Crystal structure of the hypothetical protein TA1238 from Thermoplasma acidophilum: a new type of helical super-bundle. J. Struct. Funct. Genomics 5:231–240.[PubMed] [CrossRef]
230. Santander, P. J., Y. Kajiwara, H. J. Williams, and A. I. Scott. 2006. Structural characterization of novel cobalt corrinoids synthesized by enzymes of the vitamin B12 anaerobic pathway. Bioorg. Med. Chem. 14:724–731.[PubMed] [CrossRef]
231. Santander, P. J., C. A. Roessner, N. J. Stolowich, M. T. Holderman, and A. I. Scott. 1997. How corrinoids are synthesized without oxygen: nature’s first pathway to vitamin B12. Chem. Biol. 4:659–666.[PubMed] [CrossRef]
232. Saraste, M., P. R. Sibbald, and A. Wittinghofer. 1990. The P-loop: a common motif in ATP- and GTP-binding proteins. Trends Biochem. Sci. 15:430–434.[PubMed] [CrossRef]
233. Sauve, A. A., and V. L. Schramm. 2004. SIR2: the biochemical mechanism of NAD(+)-dependent protein deacetylation and ADP-ribosyl enzyme intermediates. Curr. Med. Chem. 11:807–826.[PubMed] [CrossRef]
234. Schauer, S., S. Chaturvedi, L. Randau, J. Moser, M. Kitabatake, S. Lorenz, E. Verkamp, W. D. Schubert, T. Nakayashiki, M. Murai, K. Wall, H. U. Thomann, D. W. Heinz, H. Inokuchi, D. Soll, and D. Jahn. 2002. Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate. J. Biol. Chem. 277:48657–48663.[PubMed] [CrossRef]
235. Schmid, M. F., A. M. Paredes, H. A. Khant, F. Soyer, H. C. Aldrich, W. Chiu, and J. M. Shively. 2006. Structure of Halothiobacillus neapolitanus carboxysomes by cryo-electron tomography. J. Mol. Biol. 364:526–535.[PubMed] [CrossRef]
236. Schubert, H. L., R. M. Blumenthal, and X. Cheng. 2003. Many paths to methyltransfer: a chronicle of convergence. Trends Biochem. Sci. 28:329–335.[PubMed] [CrossRef]
237. Schubert, H. L., and C. P. Hill. 2006. Structure of ATP-bound human ATP:cobalamin adenosyltransferase. Biochemistry 45:15188–15196.[PubMed] [CrossRef]
238. Schubert, H. L., E. Raux, A. A. Brindley, H. K. Leech, K. S. Wilson, C. P. Hill, and M. J. Warren. 2002. The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase. EMBO J. 21:2068–2075.[PubMed] [CrossRef]
239. Schubert, H. L., E. Raux, K. S. Wilson, and M. J. Warren. 1999. Common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis. Biochemistry 38:10660–10669.[PubMed] [CrossRef]
240. Schubert, H. L., K. S. Wilson, E. Raux, S. C. Woodcock, and M. J. Warren. 1998. The X-ray structure of a cobalamin biosynthetic enzyme, cobalt-precorrin-4 methyltransferase. Nat. Struct. Biol. 5:585–592.[PubMed] [CrossRef]
241. Scott, A. I. 2003. Discovering nature’s diverse pathways to vitamin B12: a 35-year odyssey. J. Org. Chem. 68:2529–2539.[PubMed] [CrossRef]
242. Scott, A. I., and C. A. Roessner. 2002. Biosynthesis of cobalamin (vitamin B12). Biochem. Soc. Trans. 30:613–620.[PubMed] [CrossRef]
243. Sheppard, D. E., J. T. Penrod, T. Bobik, E. Kofoid, and J. R. Roth. 2004. Evidence that a B12-adenosyl transferase is encoded within the ethanolamine operon of Salmonella enterica. J. Bacteriol. 186:7635–7644.[PubMed] [CrossRef]
244. Sheppard, D. E., and J. R. Roth. 1994. A rationale for autoinduction of a transcriptional activator: ethanolamine ammonia-lyase (EutBC) and the operon activator (EutR) compete for adenosyl-cobalamin in Salmonella typhimurium. J. Bacteriol. 176:1287–1296.[PubMed]
245. Shiang, J. J., A. G. Cole, R. J. Sension, K. Hang, Y. Weng, J. S. Trommel, L. G. Marzilli, and T. Lian. 2006. Ultrafast excited-state dynamics in vitamin B12 and related cob(III)alamins. J. Am. Chem. Soc. 128:801–808.[PubMed] [CrossRef]
246. Shipman, L. W., D. Li, C. A. Roessner, A. I. Scott, and J. C. Sacchettini. 2001. Crystal structure of precorrin-8x methyl mutase. Structure 9:587–596.[PubMed] [CrossRef]
247. Shively, J. M., E. Bock, K. Westphal, and G. C. Cannon. 1977. Icosahedral inclusions (carboxysomes) of Nitrobacter agilis. J. Bacteriol. 132:673–635.[PubMed]
248. Shively, J. M., G. L. Decker, and J. W. Greenawalt. 1970. Comparative ultrastructure of the thiobacilli. J. Bacteriol. 101:618–627.[PubMed]
249. Smith, A. E., and R. G. Matthews. 2000. Protonation state of methyltetrahydrofolate in a binary complex with cobalamin-dependent methionine synthase. Biochemistry 39:13880–13890.[PubMed] [CrossRef]
250. Smith, B. C., and J. M. Denu. 2006. Sir2 protein deacetylases: evidence for chemical intermediates and functions of a conserved histidine. Biochemistry 45:272–282.[PubMed] [CrossRef]
251. Smith, C. A., and I. Rayment. 1996. Active site comparisons highlight structural similarities between myosin and other P-loop proteins. Biophys. J. 70:1590–1602.[PubMed] [CrossRef]
252. So, A. K., G. S. Espie, E. B. Williams, J. M. Shively, S. Heinhorst, and G. C. Cannon. 2004. A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell. J. Bacteriol. 186:623–630.[PubMed] [CrossRef]
253. Spencer, J. B., N. J. Stolowich, C. A. Roessner, and A. I. Scott. 1993. The Escherichia coli cysG gene encodes the multifunctional protein, siroheme synthase. FEBS Lett. 335:57–60.[PubMed] [CrossRef]
254. Spencer, P., and P. M. Jordan. 1994. Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli. Biochem. J. 300:373–381.[PubMed]
255. Spencer, P., and P. M. Jordan. 1993. Purification and characterization of 5-aminolaevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain. Biochem. J. 290:279–287.[PubMed]
256. Spencer, P., N. J. Stolowich, L. W. Sumner, and A. I. Scott. 1998. Definition of the redox states of cobalt-precorrinoids: investigation of the substrate and redox specificity of CbiL from Salmonella typhimurium. Biochemistry 37:14917–14927.[PubMed] [CrossRef]
257. Stabler, S. P. 1999. B12 and nutrition. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York, NY.
258. Starai, V. J., I. Celic, R. N. Cole, J. D. Boeke, and J. C. Escalante-Semerena. 2002. Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science 298:2390–2392.[PubMed] [CrossRef]
259. Starai, V. J., H. Takahashi, J. D. Boeke, and J. C. Escalante-Semerena. 2003. Short-chain fatty acid activation by acyl-coenzyme A synthetases requires SIR2 protein function in Salmonella enterica and Saccharomyces cerevisiae. Genetics 163:545–555.[PubMed]
260. Stark, W. M., C. J. Hawker, G. J. Hart, A. Philippides, P. M. Petersen, J. D. Lewis, F. J. Leeper, and A. R. Battersby. 1993. Biosynthesis of porphyrins and related macrocycles. 40. Synthesis of a spiro-lactam related to the proposed spir-intermediate for porphyrin biosynthesis: inhibition of cosynthetase. J. Chem. Soc. Perkin Trans. 1 23:2875–2892. [CrossRef]
261. Stevens, R. V. 1982. The total synthesis of vitamin B12, p. 169–200. In D. Dolphin (ed.), B12, vol. 1. John Wiley & Sons, Inc., New York, NY.
262. Stich, T. A., A. J. Brooks, N. R. Buan, and T. C. Brunold. 2003. Spectroscopic and computational studies of Co3+-corrinoids: spectral and electronic properties of the B12 cofactors and biologically relevant precursors. J. Am. Chem. Soc. 125:5897–5914.[PubMed] [CrossRef]
263. Stich, T. A., N. R. Buan, and T. C. Brunold. 2004. Spectroscopic and computational studies of Co2+ corrinoids: spectral and electronic properties of the biologically relevant base-on and base-off forms of Co2+ cobalamin. J. Am. Chem. Soc. 126:9735–9749.[PubMed] [CrossRef]
264. Stich, T. A., N. R. Buan, J. C. Escalante-Semerena, and T. C. Brunold. 2005. Spectroscopic and computational studies of the ATP:corrinoid adenosyltransferase (CobA) from Salmonella enterica: insights into the mechanism of adenosylcobalamin biosynthesis. J. Am. Chem. Soc. 127:8710–8719.[PubMed] [CrossRef]
265. St. Maurice, M., P. E. Mera, M. P. Taranto, F. Sesma, J. C. Escalante-Semerena, and I. Rayment. 2007. Structural characterization of the active site of the PduO-type ATP:co(I)rrinoid adenosyltransferase from Lactobacillus reuteri. J. Biol. Chem. 282:2596–2605.[PubMed] [CrossRef]
266. Stojiljkovic, I., A. J. Baumler, and F. Heffron. 1995. Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J. Bacteriol. 177:1357–1366.[PubMed]
267. Stroupe, M. E., H. K. Leech, D. S. Daniels, M. J. Warren, and E. D. Getzoff. 2003. CysG structure reveals tetrapyrrole-binding features and novel regulation of siroheme biosynthesis. Nat. Struct. Biol. 10:1064–1073.[PubMed] [CrossRef]
268. Stupperich, E., H. J. Eisinger, and B. Kräutler. 1988. Diversity of corrinoids in acetogenic bacteria. p-Cresolylcobamide from Sporomusa ovata, 5-methoxy-6-methylbenzimidazolylcobamide from Clostridium formicoaceticum and vitamin B12 from Acetobacterium woodii. Eur. J. Biochem. 172:459–464.[PubMed] [CrossRef]
269. Stupperich, E., H. J. Eisinger, and B. Kräutler. 1989. Identification of phenolyl cobamide from the homoacetogenic bacterium Sporomusa ovata. Eur. J. Biochem. 186:657–661.[PubMed] [CrossRef]
270. Stupperich, E., I. Steiner, and H. J. Eisinger. 1987. Substitution of Coα-(5-hydroxybenzimidazolyl)cobamide (factor III) by vitamin B12 in Methanobacterium thermoautotrophicum. J. Bacteriol. 169:3076–3081.[PubMed]
271. Suh, S., and J. C. Escalante-Semerena. 1995. Purification and initial characterization of the ATP:corrinoid adenosyltransferase encoded by the cobA gene of Salmonella typhimurium. J. Bacteriol. 177:921–925.[PubMed]
272. Suh, S. J., and J. C. Escalante-Semerena. 1993. Cloning, sequencing and overexpression of cobA which encodes ATP:corrinoid adenosyltransferase in Salmonella typhimurium. Gene 129:93–97.[PubMed] [CrossRef]
273. Taurog, R. E., H. Jakubowski, and R. G. Matthews. 2006. Synergistic, random sequential binding of substrates in cobalamin-independent methionine synthase. Biochemistry 45:5083–5091.[PubMed] [CrossRef]
274. Taurog, R. E., and R. G. Matthews. 2006. Activation of methyltetrahydrofolate by cobalamin-independent methionine synthase. Biochemistry 45:5092–5102.[PubMed] [CrossRef]
275. Textor, S., V. F. Wendisch, A. A. De Graaf, U. Muller, M. I. Linder, D. Linder, and W. Buckel. 1997. Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria. Arch. Microbiol. 168:428–436.[PubMed] [CrossRef]
276. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41:100–180.[PubMed]
277. Thomas, M. G., and J. C. Escalante-Semerena. 2000. Identification of an alternative nucleoside triphosphate: 5'-deoxyadenosylcobinamide phosphate nucleotidyltransferase in Methanobacterium thermoautotrophicum ▵H. J. Bacteriol. 182:4227–4233.[PubMed] [CrossRef]
278. Thomas, M. G., T. B. Thompson, I. Rayment, and J. C. Escalante-Semerena. 2000. Analysis of the adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) enzyme of Salmonella typhimurium LT2. Identification of residue H46 as the site of guanylylation. J. Biol. Chem. 275:27376–27386.
279. Thompson, T. B., M. G. Thomas, J. C. Escalante-Semerena, and I. Rayment. 1999. Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) complexed with GMP: evidence for a substrate-induced transferase active site. Biochemistry 38:12995–13005.[PubMed] [CrossRef]
280. Thompson, T. B., M. G. Thomas, J. C. Escalante-Semerena, and I. Rayment. 1998. Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase from Salmonella typhimurium determined to 2.3 Å resolution. Biochemistry 37:7686–7695.[PubMed] [CrossRef]
281. Tobes, R., and J. L. Ramos. 2002. AraC-XylS database: a family of positive transcriptional regulators in bacteria. Nucleic Acids Res. 30:318–321.[PubMed] [CrossRef]
282. Toraya, T. 1999. Diol dehydratase and glycerol dehydratase, p. 783–809. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York, NY.
283. Toraya, T., K. Mori, T. Hara, and T. Tobimatsu. 2000. A reactivating factor for coenzyme B12-dependent diol dehydratase. Biofactors 11:105–107.[PubMed] [CrossRef]
284. Toraya, T., K. Ushio, S. Fukui, and P. C. Hogenkamp. 1977. Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme. J. Biol. Chem. 252:963–970.[PubMed]
285. Trzebiatowski, J. R., and J. C. Escalante-Semerena. 1997. Purification and characterization of CobT, the nicotinate-mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase enzyme from Salmonella typhimurium LT2. J. Biol. Chem. 272:17662–17667.[PubMed] [CrossRef]
286. Trzebiatowski, J. R., G. A. O’Toole, and J. C. Escalante-Semerena. 1994. The cobT gene of Salmonella typhimurium encodes the NaMN:5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N 1-(5-phospho-alpha-d-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin. J. Bacteriol. 176:3568–3575.[PubMed]
287. Tsang, A. W., and J. C. Escalante-Semerena. 1998. CobB, a new member of the SIR2 family of eucaryotic regulatory proteins, is required to compensate for the lack of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase activity in cobT mutants during cobalamin biosynthesis in Salmonella typhimurium LT2. J. Biol. Chem. 273:31788–31794.[PubMed] [CrossRef]
288. Verkamp, E., M. Jahn, D. Jahn, A. M. Kumar, and D. Soll. 1992. Glutamyl-tRNA reductase from Escherichia coli and Synechocystis 6803. Gene structure and expression. J. Biol. Chem. 267:8275–8280.[PubMed]
289. Vevodova, J., R. M. Graham, E. Raux, H. L. Schubert, D. I. Roper, A. A. Brindley, A. Ian Scott, C. A. Roessner, N. P. Stamford, M. Elizabeth Stroupe, E. D. Getzoff, M. J. Warren, and K. S. Wilson. 2004. Structure/function studies on a S-adenosyl-l-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis. J. Mol. Biol. 344:419–433.[PubMed] [CrossRef]
290. Vitols, E., G. A. Walker, and F. M. Huennekens. 1966. Enzymatic conversion of vitamin B-12s to a cobamide coenzyme, alpha-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide (adenosyl-B-12). J. Biol. Chem. 241:1455–1461.[PubMed]
291. Wada, K., J. Harada, Y. Yaeda, H. Tamiaki, H. Oh-Oka, and K. Fukuyama. 2007. Crystal structures of CbiL, a methyltransferase involved in anaerobic vitamin B12 biosynthesis, and CbiL in complex with S-adenosylhomocysteine: implications for the reaction mechanism. FEBS J. 274:563–573.[PubMed] [CrossRef]
292. Wang, J., N. J. Stolowich, P. J. Santander, J. H. Park, and A. I. Scott. 1996. Biosynthesis of vitamin B12: concerning the identity of the two-carbon fragment eliminated during anaerobic formation of cobyrinic acid. Proc. Natl. Acad. Sci. USA 93:14320–14322.[PubMed] [CrossRef]
293. Wang, L., M. Elliott, and T. Elliott. 1999. Conditional stability of the HemA protein (glutamyl-tRNA reductase) regulates heme biosynthesis in Salmonella typhimurium. J. Bacteriol. 181:1211–1219.[PubMed]
294. Wang, L., S. Wilson, and T. Elliott. 1999. A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium. J. Bacteriol. 181:6033–6041.[PubMed]
295. Wang, L. Y., L. Brown, M. Elliott, and T. Elliott. 1997. Regulation of heme biosynthesis in Salmonella typhimurium: activity of glutamyl-tRNA reductase (HemA) is greatly elevated during heme limitation by a mechanism which increases abundance of the protein. J. Bacteriol. 179:2907–2914.[PubMed]
296. Warren, M. J. 2006. Finding the final pieces of the vitamin B12 biosynthetic jigsaw. Proc. Natl. Acad. Sci. USA 103:4799–4800.[PubMed] [CrossRef]
297. Warren, M. J., E. L. Bolt, C. A. Roessner, A. I. Scott, J. B. Spencer, and S. C. Woodcock. 1994. Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein. Biochem. J. 302:837–844.[PubMed]
298. Warren, M. J., and P. M. Jordan. 1988. Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase. Biochemistry 27:9020–9030.[PubMed] [CrossRef]
299. Warren, M. J., E. Raux, H. L. Schubert, and J. C. Escalante-Semerena. 2002. The biosynthesis of adenosylcobalamin (vitamin B12). Nat. Prod. Rep. 19:390–412.[PubMed] [CrossRef]
300. Warren, M. J., C. A. Roessner, P. J. Santander, and A. I. Scott. 1990. The Escherichia coli cysG gene encodes S-adenosylmethionine-dependent uroporphyrinogen III methylase. Biochem. J. 265:725–729.[PubMed]
301. Warren, M. J., N. J. Stolowich, P. J. Santander, C. A. Roessner, B. A. Sowa, and A. I. Scott. 1990. Enzymatic synthesis of dihydrosirohydrochlorin (precorrin-2) and of a novel pyrrocorphin by uroporphyrinogen III methylase. FEBS Lett. 261:76–80.[PubMed] [CrossRef]
302. Williams, L., V. Fresquet, P. J. Santander, and F. M. Raushel. 2007. The multiple amidation reactions catalyzed by cobyric acid synthetase from Salmonella typhimurium are sequential and dissociative. J. Am. Chem. Soc. 129:294–295.[PubMed] [CrossRef]
303. Wolf, J., and R. N. Brey. 1986. Isolation and characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants. J. Bacteriol. 166:51–58.[PubMed]
304. Woodcock, S. C., E. Raux, F. Levillayer, C. Thermes, A. Rambach, and M. J. Warren. 1998. Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis. Biochem. J. 330:121–129.[PubMed]
305. Woodson, J. D., and J. C. Escalante-Semerena. 2004. CbiZ, an amidohydrolase enzyme required for salvaging the coenzyme B12 precursor cobinamide in archaea. Proc. Natl. Acad. Sci. USA 101:3591–3596.[PubMed] [CrossRef]
306. Woodson, J. D., and J. C. Escalante-Semerena. 2006. The cbiS gene of the archaeon Methanopyrus kandleri AV19 encodes a bifunctional enzyme with adenosylcobinamide amidohydrolase and α-ribazole-phosphate phosphatase activities. J. Bacteriol. 188:4227–4235.[PubMed] [CrossRef]
307. Woodson, J. D., R. F. Peck, M. P. Krebs, and J. C. Escalante-Semerena. 2003. The cobY gene of the archaeon Halobacterium sp. strain NRC-1 is required for de novo cobamide synthesis. J. Bacteriol. 185:311–316.[PubMed] [CrossRef]
308. Woodson, J. D., C. L. Zayas, and J. C. Escalante-Semerena. 2003. A new pathway for salvaging the coenzyme B12 precursor cobinamide in archaea requires cobinamide-phosphate synthase (CbiB) enzyme activity. J. Bacteriol. 185:7193–7201.[PubMed] [CrossRef]
309. Woodward, R. B. 1979. Synthetic vitamin B12, p. 37–87. In B. Zagalak and W. Friedrich (ed.), Vitamin B12. de Gruyter, Berlin, Germany.
310. Xue, Y., Z. Wei, X. Li, and W. Gong. 2006. The crystal structure of putative precorrin isomerase CbiC in cobalamin biosynthesis. J. Struct. Biol. 153:307–311.[PubMed] [CrossRef]
311. Yoshizawa, Y., K. Toyoda, H. Arai, M. Ishii, and Y. Igarashi. 2004. CO2-responsive expression and gene organization of three ribulose-1,5-bisphosphate carboxylase/oxygenase enzymes and carboxysomes in Hydrogenovibrio marinus strain MH-110. J. Bacteriol. 186:5685–5691.[PubMed] [CrossRef]
312. Zayas, C. L., K. Claas, and J. C. Escalante-Semerena. 2007. The CbiB protein of Salmonella enterica is an integral membrane protein involved in the last step of the de novo corrin ring biosynthetic pathway. J. Bacteriol. 189:7697–7708.[PubMed] [CrossRef]
313. Zayas, C. L., and J. C. Escalante-Semerena. 2007. Reassessment of the late steps of coenzyme B12 synthesis in Salmonella enterica: evidence that dephosphorylation of adenosylcobalamin-5'-phosphate by the CobC phosphatase is the last step of the pathway. J. Bacteriol. 189:2210–2218.[PubMed] [CrossRef]
314. Zhou, Z. S., K. Peariso, J. E. Penner-Hahn, and R. G. Matthews. 1999. Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli. Biochemistry 38:15915–15926.[PubMed] [CrossRef]
Module3.6.3.8
