Biosynthesis and Utilization of Adenosyl-Cobalamin (Coenzyme B12)
Chapter
47
G. A. O’TOOLE, M. R. RONDON, J. R. TRZEBIATOWSKI, S.-J. SUH, and J. C. ESCALANTE-SEMERENA
Research on adenosyl-cobalamin (Ado-CBL) biosynthesis has greatly intensified since the report by Jeter et al. in 1984 which documented that Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) was able to synthesize this important macromolecule (47). The well-characterized genetic system available in this bacterium has greatly facilitated the analysis of this complex biosynthetic pathway. A combination of genetic, molecular biological, and biochemical approaches has moved this field at a very rapid pace.
This review attempts to summarize in a concise fashion what researchers in this field have learned in the last decade about the biochemistry and regulation of the Ado-CBL biosynthetic genes in S. typhimurium and Escherichia coli. An attempt is made to integrate the numerous and valuable contributions made to this field by the research groups at Rhône Poulenc-Rhorer in Paris and at the University Chemical Laboratory in Cambridge, England, who study Ado-CBL biosynthesis in Pseudomonas denitrificans.
Ado-CBL is one of the largest biologically active nonpolymeric molecules found in nature and belongs to a class of important molecules known as cyclic tetrapyrroles, which include heme, siroheme, chlorophylls, and factor F430 (27, 29). Ado-CBL is an essential nutrient for animals. In humans, a diet deficient in cobalamin (CBL) leads to the development of pernicious anemia (37).
The coenzymatic form of CBL, Ado-CBL, is composed of four main components (Fig. 1): (i) a highly decorated corrin ring in which a cobalt atom is held by coordination bonds to the nitrogen atoms of the pyrrole rings, (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, and (iv) a nucleotide loop which joins the corrin ring to the lower ligand (16). The work leading to the identification of the chemical structure of Ado-CBL, its complete organic synthesis, and crystallographic analysis fall beyond the scope of this review. The reader is referred to reviews and recent papers on these subjects (8, 12, 33, 39, 84, 105).
CBL biosynthesis has been found only in prokaryotes, including aerobes, anaerobes, and facultative anaerobes of both the bacterial and archaeal domains (7, 44, 50, 60, 86, 88, 103, 104). This review will focus on the CBL-dependent reactions, the biosynthesis of Ado-CBL, and the regulation of the Ado-CBL biosynthetic genes in S. typhimurium.
In S. typhimurium, de novo synthesis of Ado-CBL occurs only when the bacterium is growing under anaerobic growth conditions. However, if provided with a precursor with a complete corrin ring (such as cobinamide; Fig. 1), the rest of the structures (the nucleotide loop, the lower ligand, and the upper ligand) can be synthesized both aerobically and anaerobically (20, 47). Because the corrin ring of Ado-CBL is synthesized de novo only anaerobically in S. typhimurium, the analysis of this complex biosynthetic pathway can be used as a model system with which to study major pathways that are unique to the anaerobic lifestyle of this bacterium.
E. coli has been shown to synthesize CBL if provided with the precursor cobinamide (23). Only indirect evidence that some strains of this bacterium can synthesize Ado-CBL de novo has been reported. Frey and coworkers (25) showed that Ado-CBL was required for the synthesis of queuosine, and the growth conditions used strongly suggested that the strain used for these experiments was capable of de novo synthesis of Ado-CBL. Work performed by Roth and coworkers indicates that Ado-CBL biosynthesis in E. coli may be strain specific (J. R. Roth, personal communication).
In S. typhimurium, four enzymes have been shown to require CBL. None of these functions appear to be essential for cell survival under laboratory conditions.
The terminal step in the synthesis of methionine is the methylation of homocysteine by the CBL-dependent methionine synthase (MetH; EC 2.1.1.13) (95). In this reaction, 5-methyltetrahydrofolate donates a methyl group to cob(I)alamin to generate methyl-CBL, which in turn donates the methyl group to homocysteine (6).
The crystal structure of the CBL-binding domain of MetH from E. coli has recently been solved to 3-Å (0.3-nm) resolution. Information garnered from this structure has begun to provide insight into the interactions of CBL with CBL-binding proteins, including residues important for interactions with the corrin ring and the nucleotide loop of CBL (17).
The methylation of homocysteine to yield methionine can also be catalyzed by the CBL-independent enzyme MetE (EC 4.2.99.10). metE mutants are forced to use the CBL-dependent MetH enzyme in the final step of methionine synthesis. The use of metE mutants allows for a convenient (albeit indirect) assessment of the CBL biosynthetic pathway, since metE mutants with a defective CBL biosynthetic pathway are identified as methionine auxotrophs on minimal medium (47).
Ethanolamine can serve as sole carbon, energy, and nitrogen source for S. typhimurium (13, 77, 85). The enzyme ethanolamine ammonia-lyase requires Ado-CBL as a coenzyme to catalyze the first step in ethanolamine catabolism, i.e., the generation of acetaldehyde and ammonia (13). This enzyme is encoded by the eutBC genes of the eut (ethanolamine utilization) operon located at min 50 of the linkage map (22, 77, 78). Expression of the eut operon requires ethanolamine, Ado-CBL, and the positive regulatory protein EutR (78). Growth of S. typhimurium on ethanolamine as a carbon/energy or nitrogen source serves as an assay for the synthesis of Ado-CBL.
1,2-Propanediol (1,2-PDL) serves as a carbon and energy source for S. typhimurium but apparently only under aerobic growth conditions (46). 1,2-PDL dehydratase is an Ado-CBL-dependent enzyme which catalyzes the dehydration of 1,2-PDL to form propionaldehyde (70). In S. typhimurium, this enzyme is encoded by the pdu (propanediol utilization) genes located upstream of the Ado-CBL biosynthetic operon at min 41. Unlike expression of the eut operon, expression of the pdu operon requires 1,2-PDL but does not appear to require Ado-CBL (46).
Queuosine is a modified base found in four tRNAs: tRNAAsp, tRNAAsn, tRNAHis, and tRNATyr (25, 64). The synthesis of queuosine from epoxyqueuosine is catalyzed by an uncharacterized Ado-CBL-dependent enzyme (25). The function of this modified base is unknown.
S. typhimurium mutants unable to synthesize CBL were shown to carry lesions located at min 41 of the linkage map. These mutants were initially classified by their nutritional behavior (47) (Fig. 2). The nucleotide sequence of the Ado-CBL biosynthetic operon at min 41 has been determined in its entirety and shown to be 17 kb long, containing 20 putative genes (79). These genes are organized in a single operon and can be separated into two groups (Fig. 3): (i) genes involved in corrin ring synthesis, designated cbi (the operon contains 17 cbi genes), and (ii) genes involved in nucleotide loop assembly, designated cob (the operon contains 3 cob genes).
Genetic evidence and the analysis of the DNA sequence suggests that the genes in the Ado-CBL biosynthetic operon are cotranscribed from a single promoter. Additionally, the translation of some of the genes may be coupled (11, 76, 79). Genetic evidence supports the existence of two nonregulated promoters within the operon (Fig. 3) (18, 76).
Genes involved in Ado-CBL biosynthesis have been located outside the Ado-CBL biosynthetic operon. These genes are cobA (min 34) (20), cobB (min 25) (A. W. Tsang and J. C. Escalante-Semerena, unpublished results), cobC (min 14) (69), cobD (min 14) (34), and cysG (min 72) (61) (Fig. 2). The functions of these gene products are discussed below.
Only the btuR gene has been documented to be involved in Ado-CBL biosynthesis in E. coli (see below) (20, 58, 92). Homologs of the S. typhimurium cobU, cobS, cobT, and cobC genes have also been identified in this bacterium (14, 96; S. Addinali and W. Donachie, personal communication [accession number U23163]; C. M. Collins, D. M. Gutman, and J. Isaza, unpublished data [accession number L25054]).
The de novo biosynthesis of Ado-CBL can be divided into four parts: (i) synthesis of the adenosylated corrin ring, (ii) synthesis of the lower (Coα) ligand base, (iii) synthesis of the nucleotide loop, that is, joining of the corrin ring to the lower ligand, and (iv) attachment of the upper (Coβ) ligand. This section covers what is currently known about the biosynthesis of Ado-CBL in S. typhimurium.
In S. typhimurium, adenosyl-cobinamide (Ado-cobinamide) has been proposed to be the end product of the corrin ring biosynthetic pathway, and interestingly, this pathway is functional only under anaerobic growth conditions (20, 47, 48). Recent studies suggest that oxygen lability of one or more enzymes or intermediates prevents S. typhimurium from synthesizing the corrin ring aerobically (76).
Many of the genes required for Ado-cobinamide biosynthesis are located at min 41 and designated cbi (79). At least three genes involved in Ado-cobinamide synthesis, cysG (48, 51), cobA (20), and cobD (34), are located away from min 41.
Synthesis of Precorrin-3A, the Committed Intermediate in Ado-CBL Biosynthesis.
The genes involved in the corrin ring biosynthetic pathway are shown in Fig. 4. The first steps of Ado-CBL biosynthesis, the steps leading to synthesis of uroporphyrinogen III, are shared with heme and siroheme biosynthesis (for reviews of these topics, see chapters 31 and 49). Uroporphyrinogen III is the last compound which is common to the Ado-CBL, heme, and siroheme biosynthetic pathways.
The genes and gene products required for the conversion of uroporphyrinogen III to precorrin-3A in S. typhimurium have been identified and characterized. The CysG protein catalyzes the methylation of uroporphyrinogen III at C-2 and C-7 of the corrin ring (Fig. 1) in S-adenosylmethionine-dependent reactions to generate precorrin-2 (82, 101). In addition to its S-adenosylmethionine-dependent methylase activity, CysG has NAD+-dependent dehydrogenase and ferrochelatase activities needed for the synthesis of siroheme from sirohydrochlorin (also known as factor II, the oxidized form of precorrin-2) (82, 101, 102). Therefore, precorrin-2 is the last intermediate common to siroheme and Ado-CBL biosynthesis. The methylation of precorrin-2 at the C-20 position of the corrin ring by the CbiL protein yields precorrin-3A, the committed intermediate in Ado-CBL biosynthesis (75, 82).
Corrin Ring Synthesis after Precorrin-3A.
The intermediates and enzymes required for the conversion of precorrin-3A to the complete corrin ring (Ado-cobinamide) in S. typhimurium have not been characterized. However, we can begin to construct a pathway based on the DNA sequence similarity of corrin ring biosynthetic genes of S. typhimurium to genes of known function in P. denitrificans.
Figure 4 shows the pathway for corrin ring biosynthesis in P. denitrificans. For the sake of saving space, many of the original references on the work in P. denitrificans are not included in this review. The elegant studies leading to the elucidation of this pathway in P. denitrificans are reviewed by Blanche et al. (10). Also shown in Fig. 4 are the genes involved in corrin ring synthesis that are conserved between S. typhimurium and P. denitrificans. Homologs of 12 of 17 cob genes of P. denitrificans have been identified in S. typhimurium.
Comparison of the Corrin Ring Biosynthetic Pathways of S. typhimurium and P. denitrificans.
Despite the conservation of many genes, the corrin ring biosynthetic pathways in S. typhimurium and P. denitrificans may be markedly different. First, it is possible that S. typhimurium, like Propionibacterium freudenreichii (formerly Propionibacterium shermanii) (62), performs cobalt insertion early in the corrin ring biosynthetic pathway rather than late like in P. denitrificans. In P. denitrificans, the substrate for cobalt insertion, hydrogenobyrinic acid a,c-diamide, is only three steps removed from Ado-cobinamide (10). Spencer et al. (82) have suggested that in S. typhimurium, factor III (the oxidized form of precorrin-3A) may be the substrate for cobalt insertion in a reaction catalyzed by CysG.
Second, the contraction of the precorrin macrocycle (i.e., elimination of C-20) was reported to require molecular oxygen in P. denitrificans (81). Recall that the de novo synthesis of the corrin ring in S. typhimurium occurs only anoxically, making the requirement for molecular oxygen unlikely. Further work will be required to determine whether different pathways have evolved in these organisms (perhaps in response to different environmental pressures) to synthesize this complex molecule.
Cobalt Transport.
In S. typhimurium, cobalt ions are reportedly transported by the magnesium transport system, and the affinity of this system for cobalt is relatively low (38). Roth et al. (79) suggested that three S. typhimurium genes, cbiN, cbiQ, and cbiO, are involved in what may be a high-affinity cobalt transport system. This assignment is based on sequence homology, but at present, there is no experimental evidence to support this hypothesis.
In S. typhimurium, adenosylation of corrinoids is a key process both for the de novo synthesis of the corrin ring and for the assimilation of exogenous incomplete and complete corrinoids (20). The results of genetic experiments suggested that in S. typhimurium, the biosynthesis of Ado-CBL proceeds via adenosylated intermediates. Furthermore, before exogenous nonadenosylated corrinoids can be converted to Ado-CBL, they must be adenosylated (20, 67).
Adenosylation (or alkylation) of corrinoids proceeds in three steps (9, 97, 100): (i) reduction of Co(III) to Co(II), (ii) reduction of Co(II) to Co(I), and (iii) transfer of the 5'-deoxyadenosine moiety from ATP to the reduced corrinoid.
Reduction of Co(III) to Co(I).
In E. coli, two separate reductases catalyze the one-electron reductions of CBL required for the function of methionine synthase (MetH) (30, 43). In vitro studies showed that flavodoxin (the product of the fldA gene at min 15.9) can catalyze the reduction of cob(II)alamin to cob(I)alamin during the reductive activation of methionine synthase (6, 24, 66). Whether this flavodoxin participates in CBL reduction in vivo remains to be determined. Enzymes that catalyze the reduction of cob(III)alamin to cob(I)alamin have also been partially purified from Clostridium tetanomorphum (97, 100).
Blanche et al. (10) have described the purification of an NADH-dependent flavoprotein which can catalyze the reduction of Co(II) to Co(I) in P. denitrificans. This enzyme can reduce intermediates in the synthesis of Ado-cobinamide. The gene encoding this activity in P. denitrificans has not been identified. To date, no enzyme with this activity has been identified in S. typhimurium.
Transfer of the Adenosyl Moiety of ATP to Reduced Corrinoids by the CobA Protein.
The enzymatic reduction of cob(II)alamin to cob(I)alamin is thermodynamically unfavorable unless the product of the reaction [i.e., cob(I)alamin] is removed by alkylation or adenosylation (54, 55). In S. typhimurium, this last step in the corrinoid adenosylation pathway is catalyzed by the CobA protein. CobA uses ATP as the donor of the 5'-deoxyadenosyl moiety to cob(I)alamin to generate a covalent carbon-cobalt bond. CobA can also catalyze the synthesis of Ado-cobinamide from cob(I)inamide (93).
The phenotype of cobA mutants is consistent with a role in adenosylation. cobA mutants are blocked in the adenosylation of the de novo biosynthetic intermediate of the corrin ring and exogenous (complete and incomplete) corrinoids (20). The corrin ring biosynthetic intermediate that serves as the substrate for CobA has not yet been identified.
BtuR Is the Adenosyltransferase of E. coli.
BtuR and CobA have a high degree of amino acid sequence identity. Furthermore, a plasmid carrying the wild-type allele of btuR was shown to complement an S. typhimurium cobA mutant, indicating that these two proteins are functionally equivalent (92). Given the documented function of CobA as the ATP:corrinoid adenosyltransferase (93), we conclude that BtuR is the adenosyltransferase enzyme of E. coli. The reported drastic reduction in the intracellular level of Ado-CBL in btuR mutants lends further support to this conclusion (58).
Interestingly, mutations in butR result in the constitutive transcription of btuB, the outer membrane protein of S. typhimurium and E. coli required for CBL transport (35, 36, 58, 59). These data suggest that Ado-CBL is involved in the regulation of this high-affinity corrinoid transport system. This system of regulation allows communication between the Ado-CBL biosynthetic genes and the CBL transport genes.
DMB Is the Lower (Coα) Ligand Synthesized by S. typhimurium.
Cobamides isolated from several genera of bacteria and archaea contain different lower ligands, such as p-cresol, phenol (87, 88, 89), 2-methylsulfinyladenylate, 2-methylsulfonyladenylate (71), 5-hydroxybenzimidazole (91), and adenylate (90). UV-visible spectra, mass spectroscopy, and nuclear magnetic resonance spectrometry data from the analysis of the cobamide synthesized by S. typhimurium revealed that 5,6-dimethylbenzimidazole (DMB) is the lower ligand of the predominant cobamide in this organism (49).
CBL-producing prokaryotes can substitute a different, exogenously provided base for the endogenously synthesized ligand (49, 91). For example, DMB auxotrophs of S. typhimurium assimilate benzimidazole without modifications, and the resulting cobamide is biologically active (49).
Models for DMB Biosynthesis.
Two different pathways have been proposed for DMB biosynthesis. In aerobic and aerotolerant anaerobic CBL producers, DMB is thought to be derived from flavin mononucleotide in a pathway involving at least one oxygenase. In support of this pathway, Propionibacterium freudenreichii, Bacillus megaterium, Nocardia rugosa, and Streptomyces sp. have been shown to incorporate radiolabeled riboflavin into DMB (2, 3, 40, 41, 42, 57).
A second pathway has been proposed for anaerobes. On the basis of radiolabeling studies performed mostly in Eubacterium limosum, the following building blocks of DMB have been proposed: glycine contributes the C-7a, C-3a, and N-3 atoms (Fig. 1) (52), methionine is the source of the two methyl groups on the benzene ring (53), formate is the precursor of the C-2 atom, and erythrose contributes the C-4, C-5, C-6, and C-7 atoms (63, 98, 99). The precursors of DMB are similar to those in purine biosynthesis, and it has been suggested that DMB may branch from the purine biosynthetic pathway (63).
Although progress has been made in identifying putative building blocks for DMB, no genes or enzymes required for DMB biosynthesis have been identified to date. A report by Renz et al. (72) proposed that DMB is synthesized via a number of intermediates, including 5-hydroxybenzimidazole and 5-hydroxy-6-methylbenzimidazole. Interestingly, a number of intermediates and side products of this proposed pathway are bases that have been shown to serve as lower ligands in other systems (49, 91). The existence of multiple intermediates in this pathway that can serve as the lower ligand of biologically active cobamides is consistent with the inability to isolate mutants blocked in DMB biosynthesis in S. typhimurium (see below).
DMB Synthesis in S. typhimurium.
. Little is known about the pathway of DMB biosynthesis in S. typhimurium. All mutants originally isolated as DMB auxotrophs have been shown to carry lesions in the cobT gene, which specifies the NaMN:DMB phosphoribosyltransferase involved in the assembly of the nucleotide loop of CBL (96) (see below). Given the DMB auxotrophy of cobT mutants, it is formally possible that CobT also plays a role in DMB biosynthesis (14, 96). However, if CobT is not involved in DMB biosynthesis, we must conclude that no mutations affecting DMB biosynthesis have been identified.
Lower-Ligand Synthesis in E. coli.
As far as we know, lower-ligand biosynthesis in E. coli has not been investigated. However, one report describes the ability of E. coli to synthesize CBL from cobinamide (23). This result suggests that E. coli is capable of synthesizing a lower ligand to the corrin ring.
Genetics of Nucleotide Loop Assembly in S. typhimurium.
The nucleotide loop bridges the lower ligand DMB and the corrin macrocycle (Fig. 1). Our current model for nucleotide loop assembly in S. typhimurium is shown in Fig. 5. S. typhimurium mutants blocked in assembly of the nucleotide loop were isolated by their inability to synthesize CBL from the precursors cobinamide and DMB (47). At least four genes are required for nucleotide loop assembly: cobU, cobS, cobT, and cobC.
The CobU Protein.
The cobU gene codes for a bifunctional Ado-cobinamide kinase, Ado-cobinamide-phosphate guanylyltransferase enzyme that catalyzes the synthesis of Ado-cobinamide-GDP. In vitro CobU efficiently utilizes Ado-cobinamide as a substrate under oxic and anoxic conditions. CobU can also utilize hydroxo-cobinamide as a substrate but does so with a rate that is 10% of that measured when Ado-cobinamide is provided. Additionally, both CobU activities are sensitive to oxidation (G. A. O’Toole and J. C. Escalante-Semerena, unpublished data). These results are consistent with genetic studies showing that CobU (i) functions optimally in the absence of oxygen, (ii) utilizes Ado-cobinamide as a substrate aerobically and anaerobically, and (iii) participates in the assimilation of nonadenosylated cobinamide only under anaerobic conditions (67).
The identification of cobU mutants responsive to exogenous hydroxo-cobinamide-GDP is consistent with the in vitro CobU activities (68). All strains responsive to hydroxo-cobinamide-GDP have been shown to carry base substitution mutations in cobU. Interestingly, a cobU deletion strain fails to respond to exogenous hydroxo-cobinamide-GDP. On the basis of this and other observations, we postulate that CobU, CobS, CobT, and CobC proteins interact and that these interactions may be crucial to their activity (O’Toole and Escalante-Semerena, unpublished data) (see below).
The CobS Protein.
The cobS gene of S. typhimurium has been shown to encode a 26-kDa protein (68) thought to have Ado-CBL synthase activity (79). On the basis of studies of the CobU, CobT, and CobC enzymes (69, 96; O’Toole and Escalante-Semerena, unpublished data), we believe the substrates of CobS to be N 1-(α-d-ribosyl)-5,6-dimethylbenzimidazole (α-ribazole) and Ado-cobinamide-GDP and its product to be Ado-CBL, not Ado-CBL-5'-phosphate.
Nonenteric organisms may utilize N 1-(5-phospho-α-d-ribosyl)-5,6-dimethylbenzimidazole (α-ribazole-5'-P) or α-ribazole as a substrate for the Ado-CBL(-5'-phosphate) synthase (10). Studies in these organisms have suggested that Ado-CBL-5'-phosphate is an intermediate in the synthesis of Ado-CBL, though there is no evidence of this compound accumulating to significant levels in S. typhimurium. Experiments in our laboratory suggest that the end product of this pathway and the predominant cobamide produced in S. typhimurium is Ado-CBL (20, 49).
The CobT Protein.
The cobT gene codes for a 38-kDa polypeptide with NaMN:DMB phosphoribosyltransferase activity that catalyzes the synthesis of α-ribazole-5'-P (68, 96). Nutritional studies strongly suggested that α-ribazole-5'-P is an intermediate in the synthesis of the nucleotide loop (96).
The phenotypes of cobT mutants are noteworthy. When the requirement for CBL biosynthesis is low (e.g., when it is determined on the basis of methionine synthesis), cobT mutants are DMB auxotrophs (see above) (96). This DMB auxotrophy was not consistent with the demonstrated role of cobT in nucleotide loop assembly. However, the existence of a substitute CobT activity facilitates the explanation of this phenotype. We believe that the DMB auxotrophy of cobT mutants is not due to a block in the DMB biosynthetic pathway but is a result of the low affinity for DMB of a second phosphoribosyltransferase that can substitute for lack of CobT function (96). However, it is formally possible that CobT participates in DMB synthesis as well as in nucleotide loop assembly (14, 96).
cobT mutants do have a clear nucleotide loop assembly phenotype under conditions in which high levels of CBL synthesis are demanded (e.g., growth on ethanolamine as the sole carbon source) (J. R. Trzebiatowski and J. C. Escalante-Semerena, unpublished data). In conclusion, cobT is involved in nucleotide loop assembly, but the cell can compensate for loss of CobT activity as long as the demand for CBL is low.
Existence of a Substitute Function(s) for CobT Activity When the Requirement for CBL Is Low.
On the basis of genetic evidence, we postulate the existence of a substitute function for CobT encoded by the cobB locus at min 25 (96; Tsang and Escalante-Semerena, unpublished data). The phenotypes of cobB mutants (determined by the methionine synthesis assay) are consistent with this gene coding for a CobT-like phosphoribosyltransferase activity. The cobB cobT + mutant has no detectable Ado-CBL biosynthetic phenotype, the cobT cobB + mutant displays a DMB auxotrophy, and the cobB cobT double mutant has a clear nucleotide loop assembly phenotype. Furthermore, the CBL biosynthetic defect of the cobB cobT mutant is corrected by exogenous α-ribazole-5'-P, suggesting that the synthesis of this compound is blocked in the double mutant (96). The lack of a CBL biosynthetic phenotype in the cobB cobT + strain suggests that CobB may not be specific for the CBL biosynthetic pathway but instead is a nonessential enzyme recruited in strains lacking CobT. This possibility is not unprecedented. An enzyme involved in purine deoxyribonucleoside catabolism, purine-2'-deoxyribonucleosidase, can transfer a deoxyribosyl moiety from deoxyribonucleosides to DMB, albeit at a relatively slow rate (83).
An alternative model consistent with the phenotypes described above is that CobB may be required for the transport of DMB into the cell. The inability to transport exogenous DMB may not allow sufficient levels of this compound to accumulate for use by any putative CobT-like phosphoribosyltransferase activities in the cell.
The CobC Protein.
Studies in Propionibacterium freudenreichii, Clostridium sticklandii, and P. denitrificans suggested the existence of a phosphatase activity responsible for the conversion of Ado-CBL-5'-phosphate and/or α-ribazole-5'-P to Ado-CBL and/or α-ribazole, respectively (10, 26, 28, 31). These activities were detected in crude cell-free systems but were not purified or characterized in any detail.
A phosphatase specific for the Ado-CBL biosynthetic pathway has been identified in S. typhimurium (69). This phosphatase is encoded by the cobC gene, which has been cloned; its sequence was found to code for a 34-kDa polypeptide. CobC shows striking similarity to phosphoglycerate mutase, acid phosphatase, and eukaryotic fructose-2,6-bisphosphatase enzymes. It was demonstrated in vitro that CobC can use α-ribazole-5'-P as a substrate to yield α-ribazole. Consistent with these results, cobC mutants are unable to assemble the nucleotide loop of Ado-CBL (69).
The cobC gene is adjacent to cobD at min 14 of the S. typhimurium linkage map. DNA sequence analysis of the cobD and cobC genes reveals that these genes are divergently transcribed and that their putative regulatory regions may overlap (69).
Metabolic Channeling and Nucleotide Loop Assembly.
The idea of metabolite channeling (21, 80, 94) is attractive with respect to Ado-CBL biosynthesis, particularly in an organism like S. typhimurium, which synthesizes relatively small amounts of this molecule. Channeling of biosynthetic intermediates from one enzyme to the next would prevent dilution of these intermediates. The direct implication of this idea is that nucleotide loop assembly enzymes may work as a biosynthetic complex. We have preliminary genetic evidence consistent with this hypothesis (see above).
The study of DMB biosynthesis in other CBL-producing organisms has provided additional evidence for the hypothesis of metabolic channeling in CBL biosynthesis. Using radiolabeling studies, Lamm et al. (52) showed that exogenous DMB was incorporated into CBL in a nonstereospecific manner. However, when they demanded the synthesis of DMB from radiolabeled precursors, they found that DMB was attached to CBL in a stereospecific manner (52). These data suggested that DMB was not released from the enzyme that synthesized it but was passed directly to the next enzyme(s) in its biosynthetic pathway.
Nucleotide Loop Assembly in E. coli.
The only information on nucleotide loop assembly functions in E. coli reported to date consists of the identification of homologs to the S. typhimurium cobU, cobS, cobT, and cobC genes (14; Addinati and Donachie, personal communication; Collins et al., unpublished data).
Expression of the Ado-CBL biosynthetic operon at min 41 is modulated by the effector 1,2-PDL, the redox state and energy level of the cell, and the end product of the pathway.
Our laboratory discovered that 1,2-PDL positively affects transcription of the Ado-CBL biosynthetic operon at min 41 under aerobic and anaerobic conditions (76). These are the only conditions in which significant aerobic expression of this operon has been observed.
Mutations affecting 1,2-PDL-mediated expression of the Ado-CBL biosynthetic operon and pdu were mapped between these two operons (Fig. 6). These mutations define a new locus called poc, for propanediol control. DNA sequence of the poc locus identified two open reading frames, pocR and pduF (11, 15, 76, 79; M. R. Rondon and J. C. Escalante-Semerena, unpublished data).
The PocR Protein Is a Transcriptional Activator.
The pocR gene encodes a putative regulatory protein with a deduced amino acid sequence showing homology to the AraC family of transcriptional regulators (32, 79). The 1,2-PDL-dependent binding of PocR to the Ado-CBL biosynthetic operon regulatory region has been documented in vitro (Rondon and Escalante-Semerena, unpublished data). PocR may bind to multiple sites upstream of the Ado-CBL biosynthetic operon. Furthermore, the PocR binding site may overlap the regulatory region required for anaerobic expression of the Ado-CBL biosynthetic operon (73; Rondon and Escalante-Semerena, unpublished data).
Expression of the pocR gene is regulated aerobically and anaerobically. Aerobically, pocR gene expression is induced about threefold in response to 1,2-PDL (1, 11). Induction of pocR transcription by 1,2-PDL requires the wild-type PocR protein (11). This induction also requires the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP). Anaerobically, pocR gene expression is also induced about threefold in response to 1,2-PDL (1, 11). Under these conditions both ArcA and ArcB, members of the family of two-component regulatory systems (45), and CRP-cAMP play a role in induction (4).
The PduF Protein Is Likely a 1,2-PDL Facilitator.
Upstream of pocR lies the pduF gene, encoding a putative 1,2-PDL transport facilitator. This assignment was made on the basis of the similarity of the pduF gene to glpF, the glycerol facilitator protein of E. coli (15).
Expression of the Ado-CBL biosynthetic operon is induced when the cells are grown under anaerobic conditions (19). Higher levels of induction are correlated with the presence of an external electron acceptor exhibiting a lower midpoint potential; this has been termed redox regulation (5, 19). These and other data suggest that it is not the absence of oxygen that induces expression but rather that it is the redox conditions within the cell that seem to be important.
The involvement of the ArcA-ArcB regulatory system in anaerobic expression of the Ado-CBL biosynthetic operon was first reported by Andersson (4). As this induction is independent of PocR, ArcA may act directly at the promoter of the Ado-CBL biosynthetic operon (4).
Expression of the Ado-CBL biosynthetic operon is higher when cells are grown on a poor carbon source such as succinate than when they are grown on glucose (11, 19, 76). This observation is true under both aerobic and anaerobic conditions. Genetic evidence suggests a role for CRP-cAMP in this regulation (1, 19).
Expression of the Ado-CBL biosynthetic operon is repressed by the end product of the pathway, Ado-CBL (20, 74). This effect is thought to be at the posttranscriptional level. The cbi-cob transcript contains a 5' untranslated leader region of 464 nucleotides. Point mutations resulting in a loss of repression by Ado-CBL lie in this region, suggesting that the 5' untranslated region may be involved in Ado-CBL repression. A similar posttranscriptional regulatory mechanism may control expression of the E. coli (and possibly S. typhimurium) btuB gene, which is required for the transport of corrinoids (59, 74). The mechanism of Ado-CBL repression and the role of the conserved sequence are not yet understood.
Expression of the Ado-CBL biosynthetic operon, pocR, and that of pdu are induced under similar conditions, i.e., by 1,2-PDL via PocR, low redox conditions via ArcA-ArcB, and growth on poor carbon sources via CRP-cAMP. One explanation for the coregulation of the Ado-CBL biosynthetic operon and pdu is outlined below.
The 1,2-PDL-mediated induction of the Ado-CBL biosynthetic operon and pdu occurs maximally under anaerobic growth conditions and when cells are growing on a poor carbon source. Because Ado-CBL is required for the first enzyme in the pathway of 1,2-PDL degradation, this coregulation appears to make sense. However, Ado-CBL synthesis occurs only during anaerobic growth, whereas 1,2-PDL can serve as the carbon and energy source only under aerobic conditions (46, 66; Rondon and Escalante-Semerena, unpublished data). This paradox suggests that there may be conditions of reduced oxygen concentration at which both processes could occur, as has been shown recently (1). Alternatively, 1,2-PDL can be produced anaerobically (56, 65) as a result of fucose or rhamnose utilization. 1,2-PDL metabolism under these conditions could provide the cell with some unidentified benefit, despite the fact that 1,2-PDL cannot support growth anaerobically as the sole carbon source.
Finally, it is still not clear whether transcription factors such as CRP-cAMP and ArcA-ArcB act directly at the Ado-CBL biosynthetic operon promoter. These factors may modulate PocR expression, which in turn exerts its effects on the Ado-CBL biosynthetic operon promoter.
Regulation of the cobA and the cobD genes has been examined. None of the effectors that regulate expression of the main Ado-CBL biosynthetic operon (1,2-PDL, redox, carbon source, and Ado-CBL) have a significant effect on the transcription of these genes (20, 34; Rondon and Escalante-Semerena, unpublished data).
This work was supported in part by PHS grant GM40313 and USDA Hatch grant WIS3201 to J.C.E.-S.
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