Biosynthesis of the Aromatic Amino Acids
JAMES PITTARD* AND JI YANG
[SECTION EDITOR: WES HATFIELD]
Revised version posted November 7, 2008
(replaces November 15, 2004 archived version)
Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia
*Mailing address: Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia. Phone: (61) 3 8344 5696, Fax: (61) 3 9347 1540, E-mail:
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The pathway of biosynthesis of the aromatic amino acids is shown, for convenience, in three parts (Fig. 1, 2, and 3). Figure 1 shows the "common pathway" leading to the synthesis of the branch point compound chorismate, and Figure 2 shows the two terminal pathways in which chorismate is converted to phenylalanine and tyrosine, respectively. Figure 3 outlines the terminal pathway to tryptophan biosynthesis. The common pathway has sometimes been referred to as the shikimate pathway. An additional four terminal pathways lead from chorismate to the so-called aromatic vitamins folate, ubiquinone, menaquinone, and enterochelin, but these are not considered here.
Identification of the various intermediates in the aromatic pathway was completed by the early 1960s. As with other pathways, studies of auxotrophic mutants were of key importance. The first intermediate to be identified was shikimate; its position in the common pathway was established when Davis (86) showed that this compound alone of 55 aromatic and hydroaromatic compounds tested could fulfill the requirement of certain aromatic auxotrophs of Escherichia coli for tyrosine, phenylalanine, and tryptophan. Other mutants, which failed to respond to shikimate, accumulated this compound in their culture fluids. In the same study, it was established that anthranilate could be substituted for tryptophan, phenylpyruvate could be substituted for phenylalanine, and under appropriate conditions, 4-hydroxyphenyl pyruvate could be substituted for tyrosine. The accumulation by auxotrophs of intermediates preceding the blocked reaction led to the identification of 3-dehydroquinate (DHQ), 3-dehydroshikimate, and shikimate 3-phosphate, all intermediates in the common pathway (90, 284, 340, 342).
The identification of a phosphorylated compound (shikimate 3-phosphate) in the culture fluids was an exception to the general experience with other phosphorylated intermediates, in which the compounds that accumulated in culture fluids were predominantly in the dephosphorylated form. Such was the case with enol-pyruvyl shikimate (EPS) and the intermediates in the tryptophan pathway, indoleglycerol (InG) and 1-(O-carboxyphenylamino)-1-deoxyribulose (89, 100, 124, 276). Neither the phosphorylated intermediates nor their dephosphorylated derivatives serve as growth factors for any mutant blocked prior to their formation in a pathway, since the phosphorylated compounds cannot enter the cell, and the dephosphorylated derivatives are generally not true intermediates. Shikimate is a notable exception. The branch point compound chorismate fails to act as a growth factor, presumably because it is not able to enter the cell.
The early reactions in the common pathway were more difficult to establish. A mutant strain of E. coli that accumulated shikimic acid was grown on glucose variously labeled with 14C. The shikimate was isolated and chemically degraded to establish the distribution of specific carbon atoms of glucose in the shikimate molecule. The results showed that three of the carbon atoms of shikimate were derived from glycolysis and four were derived from the pentose phosphate pathway (314).
The nature of these precursor compounds was established when erythrose 4-phosphate became available (23) and it was demonstrated that cell extracts of E. coli could convert erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) to DHQ. Furthermore, on fractionation, an enzyme preparation could be obtained that converted E4P and PEP to 2-keto-3-deoxy-d-arabo-heptonic acid 7-phosphate (312). This compound was later to be termed 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP).
The identification of chorismate, the branch point compound, required the use of mutant strains blocked in each of the three terminal pathways. A strain of Aerobacter aerogenes (62-1) that was blocked in the second reaction of the tryptophan pathway, and the first reactions of both the phenylalanine and the tyrosine pathways, was isolated. Extracts of this strain were able to convert shikimate to anthranilate. When, however, glutamine was omitted from the reaction mixture, a new compound was formed. This compound could be extracted from the reaction mixture and used as a substrate for anthranilate production by extracts of a mutant blocked after the EPS phosphate (EPSP) step (126). Subsequent studies showed that the compound could be accumulated by strain 62-1 and that it could be converted enzymatically into anthranilate, prephenate, 4-hydroxyphenylpyruvate, phenylpyruvate, and 4-hydroxybenzoate. The compound was called chorismic acid (123, 126). After the structure of chorismate had been established, it was shown that it could be formed from EPSP by cell extracts of E. coli (234). The position of EPSP in the pathway was further supported by the demonstration that cell extracts produced EPSP from shikimate 3-phosphate and PEP and could convert EPSP to phenylpyruvate (204).
All of the reactions shown in Fig. 1, 2, and 3 have been confirmed in cell extracts; many of these experiments used purified enzyme preparations. Although much of the early work establishing the pathway used E. coli and, in some cases, A. aerogenes, the reactions apply equally to Salmonella enterica serovar Typhimurium. Recent interest in the pathway has been stimulated by the recognition that some of the enzymatic reactions may be excellent targets for new antimicrobial compounds (301). There has also been extensive engineering of the pathway in E. coli, in an attempt to produce commercially viable processes for the production of aromatic amino acids, aromatic vitamins, or important non-aromatic intermediates (26, 119, 255). Research has also been stimulated by the realization that the attenuation of some pathogenic strains to create potential vaccines resulted from blocks in the pathway of biosynthesis of the aromatic amino acids and vitamins (176, 321, 330). Furthermore, the role of the EPSP synthase enzyme in glyphosate sensitivity and resistance in plants (108) has drawn attention to this particular reaction and others in the pathway in the search for new herbicides.
Both E. coli and Salmonella have three isofunctional DAHP synthase enzymes (39, 50, 99, 224, 271, 294, 306). Each one is inhibited by a different amino acid, DAHP synthase (TYR) by tyrosine, DAHP synthase (PHE) by phenylalanine and DAHP synthase (TRP) by tryptophan. Whereas both DAHP synthase (PHE) and (TYR) can be inhibited to greater than 95% by the cognate amino acid, inhibition of DAHP synthase (TRP) by tryptophan does not exceed 60% (264, 272). The partial inhibition of DAHP synthase (TRP) ensures that even in the presence of all three aromatic amino acids there is a residual biosynthetic capacity able to provide enough chorismate for synthesis of the aromatic vitamins (333).
Expression of each of the genes coding for the different isoenzymes is also subject to transcriptional control. The expression of aroH [DAHP synthase (TRP)] is repressed by the TrpR protein in the presence of tryptophan, aroF [DAHP synthase (TYR)] is repressed by the TyrR protein in the presence of tyrosine and, to a lesser extent, by phenylalanine. On the other hand, the expression of aroG [DAHP synthase (PHE)] shows only small changes in response to the aromatic amino acids, but its transcription is repressed by increases in the level of TyrR protein (41, 42, 51, 85, 165, 264, 333, 334).
Throughout this chapter we have retained the genetic nomenclature, which has been used in reporting these studies in E. coli and Salmonella. Recently, it has been argued (45) that, in order to compare what is happening in a wide range of organisms, a single system of nomenclature in which aroA indicates a gene for the first reaction of the pathway, aroB the second, aroC the third, and so on, is required. Although there are clear merits in this proposal, its adoption for this chapter would only add unnecessary confusion and, hence, we have retained the published nomenclature for the genes.
The different regulation of each of these three DAHP synthases by the three aromatic amino acids was used in a strategy to recover mutants lacking each one of these activities (42, 331, 332). Each of the DAHP synthase enzymes has a requirement for divalent cations, which can be met to different degrees by a number of divalent metals, including Mn2+, Cd2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+ (318). Each of the enzymes has a high affinity for PEP (approximately 10 μM) and it has been proposed that within the cell PEP is normally bound to the enzymes. PEP has been shown to stabilize DAHP synthase during purification and storage and to protect against heat inactivation (224, 240, 294). In the absence of PEP the instability of the enzyme can be prevented by EDTA and it has been shown that, in the absence of PEP, redox metal ions that normally activate the enzyme cause oxidation of the two active-site cysteinyl residues Cys61 and Cys328 (253). The various DAHP synthases show different rates of decay when cells enter stationary phase, which may reflect differences in sensitivity to oxidation under conditions in which PEP may become limiting.
Whereas DAHP synthases (TYR) and (TRP) are both dimers, DAHP synthase (PHE) exists as a tetramer. The monomers are approximately the same size and show a great deal of sequence homology indicating that the various forms have arisen as a result of gene duplication and divergence (272). These isoenzymes in E. coli and Salmonella belong to the A1a group of DAHP synthases widely represented in gram-negative bacteria (320). The crystal structure of DAHP synthase (PHE) complexed with PEP and Pb2+ has been determined. The protein is a tetramer and each of the monomers is a (β/α)8 barrel with an N-terminal extension and a two stranded β-sheet inserted between helix α5 and strand β6. The active site and the inhibitor-binding site have been identified and shown to be separated by at least 15 Å. The inhibitor-binding site comprises a cavity that lies outside the core of the barrel. Various residues, whose substitution affects feedback inhibition in one or other of the three isoenzymes, also cluster on the outer side of the β-core of the barrel behind the β3 and β4 strands (300). Further studies involving the crystal structure of the enzyme complexed with Mn2+ and a substrate analogue 2-phosphoglycolate have revealed details of the active site of the enzyme (329). This has been further refined by using a dimeric form of the protein and high resolution (300). Studies of crystals complexed with Mn2+, phosphenolpyruvate, and phenylalanine have revealed the conformational changes transmitted from the inhibitor-binding site to the active site on the binding of phenylalanine (302). Studies of the structures of the tyrosine-inhibitable and phenylalanine-inhibitable DAHP synthase enzymes of Saccharomyces cerevisiae revealed that they were almost identical in structure to the E. coli DAHP synthase (PHE) (143). Furthermore it was established that a single amino acid residue at the base of the inhibitor-binding cavity determined whether the enzyme was inhibited by tyrosine or by phenylalanine. Although there were 61 amino acid residues that differed between the tyrosine- and phenylalanine-inhibitable enzymes, changing only one of these, a serine at the base of the inhibitor-binding cavity to a glycine, converted a phenylalanine-inhibitable enzyme into a tyrosine-inhibitable enzyme. An examination of DAHP synthase sequences from Candida, Aspergillus, Escherichia, and Haemophilus confirmed the relationship between a serine residue and phenylalanine inhibition and a glycine residue and tyrosine inhibition (143).
When cells are grown in minimal medium, DAHP synthase (PHE) is the major isoenzyme present (approximately 80%). Under conditions in which the aromatic amino acids limit growth, as in minimal medium supplemented with all amino acids except phenylalanine, tyrosine, and tryptophan, DAHP synthase (TYR) becomes the major enzyme. DAHP synthase (TRP) always remains a minor component even when expression of the gene is derepressed (95, 322).
DHQ synthase, formerly known as 5-dehydroquinate synthetase, catalyzes the conversion of DAHP to DHQ. DHQ was first identified as an intermediate in aromatic biosynthesis in 1953 (340). It was isolated from the culture fluids of an aromatic auxotroph of E. coli. The active compound had previously been shown to support the growth of mutants blocked very early in the aromatic pathway. Its structure was determined and it was shown to be chemically convertible to dehydroshikimate by heat at an acid pH (90). The enzymatic conversion of DAHP to DHQ was first reported in 1963 with a partially purified enzyme preparation from E. coli. The enzyme required both NAD+ and Co2+ for activity (313). Studies by Rotenberg and Sprinson (281, 282) with DAHP specifically labeled with tritium established that the cyclization step involves an interaction between the enol formed in phosphate elimination and the carbonyl at C-2 in an aldolase-type reaction. Kinetic isotope effects were observed at C-5, supporting a mechanism involving oxidation at C-5 by NAD. The finding that all the tritium of labeled DAHP is conserved in DHQ established that hydride transfer in the subsequent reduction of C-5 involved the same hydrogen atom that was taken from DAHP. NADH is enzyme bound and reduced the keto group at C-5 with the regeneration of NAD.
DHQ synthase has been purified to homogeneity from E. coli B and shown to be a single polypeptide with a molecular weight of 57,000. The native enzyme is a monomer, and the Km for DAHP is 33 μM (216). The structural gene for this enzyme (aroB) was cloned and sequenced; the protein was overexpressed, and its amino acid composition and amino-terminal sequence were determined. From the sequence, the monomer is deduced to be a protein of 362 amino acids with a calculated Mr of 38,800 (229).
Frost et al. (112) used gene-cloning techniques to produce high levels of DHQ synthase for purification. They purified the enzyme to homogeneity and showed it to be a monomeric protein with an Mr between 40,000 and 44,000. In microbial eukaryotes, DHQ synthase activity is carried on a pentafunctional protein called AROM, which comprises the five central enzymes of the shikimate pathway. Carpenter et al. (53) have determined the crystal structure of the DHQ synthase domain of this protein and provided much structural detail relevant to the molecular reactions catalyzed by this enzyme. The extensive homology between this domain and the DHQ synthases of E. coli and Salmonella make it likely that enzymatic mechanisms deduced from this structure also apply to these enzymes.
As with the other remaining enzymes of the common pathway (with the exception of shikimate kinase and EPSP synthase), DHQ synthase appears to be synthesized constitutively. Its synthesis is not repressed by any of the aromatic amino acids or by chorismate, nor is it induced by DAHP. The specific activity of DHQ synthase in extracts of E. coli is 25 mU/mg of protein. It has been calculated that this is about fivefold greater than would be required to meet the aromatic needs of cells growing with a doubling time of about 1 h (322). Strains of E. coli with feedback-resistant DAHP synthase (TYR) accumulate DAHP, indicating that under these conditions, DHQ synthase activity has become rate limiting (246). DHQ synthase activity is inhibited by high concentrations of tyrosine (0.6 mM or greater) (26). Unexpectedly, the aroB gene is found to be part of a single transcription unit comprising seven genes of diverse functions. Although three of these, aroB, aroK, and trpS have functions relevant to the synthesis or use of aromatic amino acids, there seems to be no particular advantage in this grouping. Detailed studies to identify transcriptional patterns under different conditions will be required before the proposed term of supraoperon is really justified (210).
DHQ dehydratase, commonly known as dehydroquinase, catalyzes a cis elimination of water to convert DHQ to 3-dehydroshikimate and to introduce the first double bond of the aromatic ring (44, 142, 325). The enzymatic reaction was first studied by Mitsuhashi and Davis (231), who partially purified the enzyme from both A. aerogenes and E. coli. They separated DHQ dehydratase from dehydroshikimate reductase (now known as shikimate dehydrogenase) and demonstrated the reversibility of the reaction. The Km for DHQ was 44 μM. Among plants, fungi, and microorganisms there are two distinct classes of dehydroquinate dehydratase enzymes, each possessing different structures and utilizing different enzymatic mechanisms (182, 296, 347). The enzymes present in E. coli and Salmonella belong to class I, whose members are exclusively involved in the synthesis of shikimate. Class II enzymes found in plants and other microorganisms are sometimes involved in biosynthetic reactions and sometimes in the catabolic pathways involving quinic acid. The DHQ dehydratase from E. coli was shown to be a simple dimeric protein with a subunit Mr of 29,000 (56). The structure of the DHQ dehydratase from S. typhi has been solved and the monomers have been shown to have an overall topology involving an eight-stranded α/β barrel (132). Important residues involved in proton abstraction and in the formation of the Schiff’s base intermediate have been identified (203). As previously mentioned for DHQ synthase, DHQ dehydratase appears to be synthesized constitutively. When E. coli and serovar Typhimurium are grown in minimal medium, DHQ dehydratase specific activities range from 50 to 100 mU/mg (128, 322).
Examination of the E. coli genome sequence leads to the prediction that the gene for dehydroquinate dehydratase, aroD, is the third gene in a transcription unit b1691, ydiB, aroD. The gene ydiB is a paralogue of aroE, the gene for shikimate dehydrogenase, and will be discussed in the next section. The function of b1691 is unknown.
The enzyme that converts 3-dehydroshikimate to shikimate was first studied in 1954 with partially purified extracts of E. coli W. The reaction was studied in the reverse direction (shikimate to dehydroshikimate) in a coupled reaction with glutathione reductase to oxidize the NADPH that is formed. The reaction depends on NADP and is specific for shikimate. Shikimate dehydrogenase activity was not detected in extracts of aromatic auxotrophs, which accumulated dehydroshikimate. The Km of the enzyme for shikimate was 55 μM (365). The reaction is stereospecific, involving transfer of hydrogen from the A side of NADPH (82). Chaudhuri and Coggins purified shikimate dehydrogenase from E. coli to homogeneity and reported that it is a monomeric protein with an Mr of 32,000 (55). The structural gene (aroE) was cloned and sequenced. The amino acid composition and amino-terminal sequence of the purified protein were determined. These results indicate that the monomer is a polypeptide of 272 amino acids with a calculated Mr of 29,380 (13). The constitutive level of this enzyme in cells grown in minimal medium is about 60 mU/mg (322).
A search of the E. coli genome sequence revealed a second gene, which encodes a protein YdiB that shares 25% sequence identity with AroE throughout its entire length and is regarded as a paralogue of AroE. Both AroE and YdiB have been overexpressed and purified, and the structures of the purified enzymes have been determined (32, 214, 225). Whereas the aroE-encoded enzyme oxidizes shikimate with NADP+, it will not function with NAD+ as cofactor, nor is it able to use quinic acid as a substrate. On the other hand, YdiB can use either NAD+ or NADP+ and can utilize either shikimic acid or quinic acid as substrates. Although the affinities of both enzymes for their respective cofactors and for shikimate are comparable, YdiB has a much lower catalytic activity and its physiological role in the cell is at the moment an enigma. Its presence in a transcription unit with aroD, which encodes the enzyme for the previous reaction in the pathway, would perhaps favor a role in the biosynthetic pathway that does not seem to be supported by the enzyme studies. The original E. coli mutant used to locate the aroE gene on the chromosome has a complete block in the pathway, requiring the aromatic vitamins as well as the aromatic amino acids for growth, but the status of YdiB in this strain has not been determined (266). The detailed studies of the structures of both AroE and YdiB have provided a great deal of information about the catalytic site and molecular transformations carried out by these enzymes (32, 225).
Shikimate kinase catalyzes the formation of shikimate 3-phosphate from shikimate and ATP. Shikimate 3-phosphate was first identified, as a possible intermediate in aromatic biosynthesis by Davis and Mingioli (89). Its chemical structure was established by Weiss and Mingioli (342). No mutants blocked in the shikimate kinase reaction were found among aromatic auxotrophs of either E. coli (266) or serovar Typhimurium (128). The reasons for this became apparent when it was shown that extracts of serovar Typhimurium yield two separable peaks of shikimate kinase activity on DEAE-cellulose (235). Using ultracentrifugation in sucrose density gradients, Berlyn and Giles (34) confirmed the observation made in Salmonella spp. and established that E. coli also had two shikimate kinase enzymes. Further study of the E. coli enzymes established that the synthesis of one of these (called shikimate kinase II because it was the second shikimate kinase to be eluted from DEAE-Sephadex) was subject to specific control involving the regulator gene tyrR. In particular, when cells were starved for tyrosine and tryptophan or when they carried an inactive tyrR regulator gene, synthesis of shikimate kinase II was derepressed about 10-fold, levels varying between 5 and 55 mU/mg.
By studying the levels of constitutive synthesis by a tyrR mutant growing at different growth rates, predictions were made about the possible chromosomal location of the aroL gene (322). This was confirmed by the use of selective F' strains and localized mutagenesis was employed to isolate a mutant lacking shikimate kinase II activity (107). The isolation of this mutant facilitated the subsequent cloning and sequencing of the aroL gene (92, 94, 230). The protein translated from the sequence has a Mr of 18,998.
The gene encoding shikimate kinase I (aroK) was identified as part of a transcription unit including aroB (208). The aroK sequence shows a 30% homology to shikimate kinase II over its entire length (345). An examination of the kinetics of the enzymatic reaction with fully purified shikimate kinase II and partially purified shikimate kinase I revealed important differences between the two enzymes. The Km of shikimate kinase II for shikimate is 200 μM. On the other hand, the Km of shikimate kinase I for shikimate appears to be in excess of 5 mM (94). This apparent low affinity of shikimate kinase I for shikimate explains why aroL mutants that lack shikimate kinase II activity will grow in minimal media only if the levels of DAHP synthase are high. Neither enzyme is inhibited by any of the end products. A further marked difference between the two enzymes is that mutants lacking aroK activity are resistant to the antibiotic Mecillinam, whereas aroL mutants show no change in Mecillinam sensitivity (327). The structures of the aroK-encoded enzyme from E. coli and shikimate kinase II from Erwinia chrysanthemi have been determined and shown to be structurally similar (190, 278).
The existence of isofunctional enzymes has been observed most frequently in the first reaction of a pathway that later branches to multiple end products, e.g., aspartokinases for lysine, methionine, and threonine (66) and DAHP synthases for the aromatic amino acids. It is also true that specific regulation of gene expression most frequently affects genes specifying the first enzyme in a pathway. It is noteworthy that, of all the genes of the common pathway of aromatic biosynthesis, only the three specifying the three DAHP synthase enzymes and the gene for shikimate kinase II are subject to control. Does this mean that, in the evolutionary past, aromatic biosynthesis started with shikimate, or does it indicate an as yet undiscovered pathway diverging from shikimate? The very low affinity of shikimate kinase I for shikimate raises the possibility that this enzyme has another major function unrelated to biosynthesis of the aromatic amino acids.
EPSP synthase converts PEP and shikimate 3-phosphate into EPSP. It was the dephosphorylated form of this compound that was first identified in culture fluids of aromatic auxotrophs blocked in the last step of the common pathway (89). Levin and Sprinson (204) partially purified the enzyme activity from E. coli and showed that the product of the reaction was EPSP. The mechanism of this reaction was studied with partially purified enzyme from Salmonella spp. and shown to involve the transfer of the enolpyruvyl grouping unchanged to the acceptor molecule (38). This reaction introduces the three-carbon fragment that is destined to become the side chain of phenylalanine and tyrosine but to be replaced in the synthesis of tryptophan. The enzyme from E. coli has been purified to homogeneity (205). The subunit Mr was estimated to be 49,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the native molecular weight was estimated to be 55,000 by gel filtration, confirming that the enzyme is a monomer.
The gene for this enzyme, aroA, has been cloned and strains carrying this gene on a multicopy plasmid have been used to produce EPSP synthase (104). The Kms for PEP and shikimate 3-phosphate are 16 and 2.5 μM, respectively. The amino-terminal sequence and the amino acid composition of the purified protein have been determined. This information, in conjunction with the nucleotide sequence of the aroA gene, indicates that the primary translation product is a polypeptide of 427 amino acids with a calculated Mr of 46,112 (103). Intensive studies of the active site and the molecular details of enzyme catalysis have been prompted by the importance of this enzyme in plants where it is the target for the herbicide glyphosate. The three-dimensional structure of the enzyme has been determined in the presence of its substrate and also in the presence of the inhibitor glyphosate (293, 316). Along with UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), EPSP synthase constitutes a small enzyme family of enol-pyruvyl transferases of considerable interest in the development of new antimicrobial agents. This has stimulated an increased interest in the mode of action of these enzymes (63, 109, 232).
An analysis of the DNA sequence of the region containing aroA, coupled with polarity studies in both E. coli and Salmonella, revealed that aroA is part of a transcription unit with serC (pdxF) in which serC (pdxF) is the first gene of the operon (155). A rho-independent terminator between serC (pdxF) and aroA results in an eight times greater amount of the single gene serC (pdxF) transcript compared with the cotranscript also including aroA. Previous reports had suggested that expression of aroA was unaffected by either of the aromatic regulators TyrR and TrpR (128, 322). However, when E. coli was grown in media containing 20 amino acids, levels of EPSP synthase were found to be repressed about fivefold (322). Subsequently, a careful examination of expression of the serC (pdxF) promoter in a variety of media and involving regulator mutants crp and lrp has shown that the serC (pdxF)-aroA operon is activated by Lrp and repressed by Crp. The combined effects of these regulators provide maximum expression in glucose minimal medium and lowest expression in rich media such as Luria broth (218).
The chorismate synthase reaction introduces a second double bond into the aromatic ring system and forms the branch point compound chorismate, which serves as the starting substrate for the three terminal pathways of aromatic amino acid biosynthesis and for the pathways to ubiquinone, menaquinone, folate, and enterochelin (123, 126). The enzyme has been partially purified from E. coli. It is oxygen sensitive and requires a reducing environment that can be established with an FADH2- and NADH-regenerating system in an anaerobic environment. Activity is inhibited by iron chelators and by high levels of Fe2+. The basis for the latter effect is unknown, but it is postulated that Fe2+ is a cofactor for chorismate synthase and that the reaction is irreversible. A tentative molecular weight for the enzyme of somewhere between 70,000 and 100,000 has been proposed (234).
The enzyme has a strict requirement for reduced FMN and, along with other chorismate synthases derived from bacteria and plants, is monofunctional using reduced FMN present in the cellular environment, whereas the bifunctional enzymes found in fungi and protozoa can directly reduce the bound cofactor FMN at the expense of NADPH (106).
There are two reports of the cloning and sequence analysis of the gene for chorismate synthase (aroC) of E. coli (54, 348). A difference between the two nucleotide sequences results in different predicted amino acid sequences for the 36 amino acids at the carboxyl end of the protein. Removal of these amino acids does not destroy enzyme activity, so the reported difference may be a true polymorphism. The calculated Mr for the monomer from E. coli is either 39,138 or 38,183, and the Mr for the monomer from serovar Typhimurium is 39,108. Gel filtration experiments show that the native enzyme is a tetramer (348). The secondary structure of the enzyme has been investigated using far-UV CD spectroscopy and is predicted to be an α/β-barrel (211). Substrate and cofactor analogues have been used to probe the mechanism of the enzymatic reaction (249). The crystal structure of chorismate synthase has been determined in two separate studies involving Streptococcus pneumoniae and Helicobacter pylori (6, 213). In both cases the enzyme is a tetramer with the monomer possessing a novel α-β-α sandwich fold. Important features of the active site and the binding of EPSP and FMN are described that would be of relevance to the closely related structures proposed for the enzyme from E. coli.
Synthesis of the enzyme is believed to be constitutive in both serovar Typhimurium and E. coli. The specific activity of chorismate synthase in cell extracts is only 10% to 20% of that of the prior enzyme, EPSP synthase (128, 322). It is puzzling that the last reaction of the pathway appears to be rate limiting, unless such a situation facilitates subsequent controls directing chorismate along the various terminal pathways. Clearly, more work needs to be done on this important enzyme and its role in vivo in aromatic biosynthesis. An analysis of the E. coli genome sequence indicates that aroC may be expressed as part of a transcription unit comprising a number of putative genes, i.e., yfcB, aroC, mepA, yfcA, b2326, b2325. No detailed studies on this transcription unit have yet been reported.
The first reaction of both the phenylalanine and the tyrosine pathways involves the conversion of chorismate to prephenate. The structure of prephenate was determined by Weiss et al. (341). This relatively unstable compound can be converted to phenylpyruvate at an acid pH resulting in some puzzling results when phenylalanine auxotrophs were first studied. Simmonds (304) described a phenylalanine auxotroph that appeared on prolonged culture to provide its own auxotrophic requirements. Davis (88) and Katagiri and Sato (178) investigated this phenomenon and suggested that the substrate of the blocked reaction was being converted chemically to a precursor in phenylalanine biosynthesis. Davis showed that the accumulated substrate is prephenate and that the phenylalanine precursor is phenylpyruvate. The enzymes that carry out the synthesis of prephenate have been referred to as chorismate mutases. Cotton and Gibson (73) showed that chromatography of cell extracts of A. aerogenes and E. coli on DEAE-cellulose gave two well-separated proteins with chorismate mutase activity. Prephenate dehydratase activity was associated with the first component to be eluted, and prephenate dehydrogenase activity was associated with the second. Prephenate dehydratase carries out the second reaction of the phenylalanine pathway, and prephenate dehydrogenase carries out the second reaction in the tyrosine pathway (73). Subsequent purification of these enzymes from E. coli has confirmed that, in each case, both activities are the product of a single bifunctional enzyme (98, 122, 159, 186, 187).
The phenylalanine enzyme is referred to as chorismate mutase-prephenate dehydratase (encoded by pheA), and the tyrosine enzyme is referred to as chorismate mutase-prephenate dehydrogenase (encoded by tyrA). Serovar Typhimurium also contains a monofunctional chorismate mutase, which is expressed in the periplasm of the cell. However, the role of this enzyme in aromatic amino acid biosynthesis is yet to be established (45).
Chorismate mutase-prephenate dehydratase is a homodimer with a subunit molecular weight of about 40,000 (21, 84, 122). A number of studies indicate that chorismate mutase-prephenate dehydratase has two independent catalytic sites (21, 91, 266). The two enzymatic activities can be inhibited differentially by chemical modifying reagents (120, 121), by phenylalanine (98), and by substrate analogues (22). Kinetic studies and the use of radioactively labeled chorismate suggest that when the reaction is carried out with purified enzyme in vitro, prephenate dissociates from the mutase site and equilibrates with the bulk medium before combining at the dehydratase site (102). Inhibition of prephenate dehydratase by phenylalanine approaches 90% in both E. coli and serovar Typhimurium, and the associated mutase activity in both cases is inhibited 55% (98, 290, 291). The use of genetic engineering to express discrete domains from the overall protein has demonstrated that both mutase and dehydratase activities are present in a polypeptide comprising residues 1 to 285, but neither activity is inhibited by phenylalanine. Polypeptides comprising residues 101 to 300 retain full dehydratase activity but lack mutase activity and fluorescence emission spectra and binding assays involving the wild-type protein indicate that residues 286 to 386 are crucial for phenylalanine binding (380). Site-directed mutagenesis coupled with the isolation of mutant proteins has identified four residues important for dehydratase activity (T278, N160, Q215, and S208) (381). Isothermal titration calorimetry, in conjunction with the study of certain selected mutant proteins, has identified two highly conserved regions, GALV (residues 309 to 312) and ESRP (residues 329 to 332), as important for phenylalanine binding and feedback inhibition (268). Earlier studies had reported that mutations located within codons 304 to 310 resulted in complete loss of feedback inhibition (242).
Chorismate mutase-prephenate dehydrogenase also forms homodimers with a subunit molecular weight of about 40,000 (159, 187). Chorismate mutase-prephenate dehydrogenase mutants that have lost dehydrogenase activity, but have retained mutase activity, have been isolated (279). Although chemical modification of the purified enzyme causes parallel loss of both activities (159, 186), tyrosine differentially inhibits prephenate dehydrogenase activity (157), NAD activates chorismate mutase activity, and chorismate stimulates prephenate dehydrogenase activity (154). Kinetic and computer simulation studies support a single active site (153). However, the identification of an inhibitor that inhibits prephenate dehydrogenase activity without affecting chorismate mutase activity may indicate discrete binding pockets within separate or partially overlapping active sites (326). Tyrosine can cause up to 95% inhibition of prephenate dehydrogenase activity and, in the presence of NAD, up to 45% inhibition of the associated chorismate mutase activity (157). Site-directed mutagenesis has been used to identify residues critical for either mutase or dehydrogenase activity. Substituting lysine 37 by glutamine destroys mutase without affecting dehydrogenase activity, whereas changing histidine 197 to asparagine destroys dehydrogenase without affecting mutase activity (59). Attempts to identify discrete domains in the T protein by engineering peptide fragments, as had been done for the P protein, indicated that neither the mutase nor the dehydrogenase activity could be expressed in a fully functional form as a discrete contiguous subregion of the T protein. Tyrosine binding and feedback inhibition could not be attributed to a structural domain separate from the mutase and dehydrogenase domains, but it was possible to show that two C-terminal sequences lacking approximately one quarter of the T protein from the N terminus had lost all mutase activity while retaining significant levels of dehydrogenase activity (57).
The gene for chorismate mutase-prephenate dehydrogenase (tyrA) is part of an operon with aroF, and its expression is coordinately regulated by TyrR protein with tyrosine as a corepressor. The expression of the gene for chorismate mutase-prephenate dehydratase (pheA) is regulated by a system of attenuation that senses the availability of charged phenylalanyl-tRNAPhe and is discussed later.
Because each of these enzymes and anthranilate synthase compete with each other for the same substrate, chorismate, it is of interest to compare the affinities of each for this substrate. The Kms of highly purified enzyme preparations are as follows. Chorismate mutase-prephenate dehydratase from E. coli has a Km for chorismate of 45 μM (98), whereas chorismate mutase-prephenate dehydrogenase from the same organism has Kms for chorismate of 92 μM and for prephenate of 50 μM (159). In contrast, the Km of anthranilate synthase for chorismate is 1.2 μM (20). This very marked difference in affinities implies that, under conditions of chorismate limitation, this compound would be preferentially directed down the tryptophan pathway rather than toward phenylalanine or tyrosine. This difference may explain why aromatic auxotrophs with incomplete blocks in any of the common pathway reactions seem able to meet their requirement for tryptophan before their requirement for phenylalanine and tyrosine (87).
The last reaction in both the phenylalanine and the tyrosine pathways involves transamination of the respective α-ketoacids, phenylpyruvate, and 4-hydroxyphenylpyruvate, with glutamate as the amino donor. Early studies on aminotransferases in E. coli indicated that there were a number of aminotransferase enzymes and that these enzymes had rather broad specificities (58, 68, 222, 223, 233, 283, 303). Phenylalanine and tyrosine could be formed by at least two enzymes, one of which was termed the aromatic aminotransferase and the other was termed the aspartate aminotransferase. Synthesis of the aromatic aminotransferase could be repressed by tyrosine, but synthesis of the aspartate aminotransferase appeared to be constitutive. There was at least one more enzyme that could function in the conversion of phenylpyruvate to phenylalanine. The analysis of this rather complex situation has been greatly assisted by the isolation of mutants lacking individual aminotransferase enzymes (117, 118, 324) coupled with the development of methods for the separation of aminotransferase activities (222, 223, 270).
Three enzymes can contribute to aromatic amino acid biosynthesis, the so-called aromatic aminotransferase coded for by tyrB, the aspartate aminotransferase coded for by aspC, and the branched-chain amino acid aminotransferase coded for by ilvE. Double mutants inactivated in both tyrB and aspC require tyrosine but not phenylalanine for growth, whereas mutants lacking all three activities require both phenylalanine and tyrosine (118). The purification of the aspartate and the aromatic aminotransferase enzymes provided an opportunity to compare their properties. This has been done by Mavrides and Orr (223), by Powell and Morrison (270), and more recently by Hayashi et al. (146). Both enzymes are homodimers with subunit molecular weights of approximately 43,000.
The aspartate aminotransferase has a much higher affinity for aspartate than does the aromatic aminotransferase. On the other hand, the affinity of the aromatic aminotransferase for phenylalanine and tyrosine and their α-ketoacids is much greater than that of the aspartate aminotransferase. The aminotransferase reaction is freely reversible and it seems that, under normal physiological conditions, phenylalanine and tyrosine syntheses are primarily carried out by the aromatic aminotransferase, the product of the tyrB gene. Only when the pools of phenylpyruvate and 4-hydroxyphenylpyruvate become very large will the aspartate aminotransferase contribute to the synthesis of these two amino acids. The converse is true when one considers the synthesis of aspartate. It is not known whether the ilvE-encoded aminotransferase is ever involved in phenylalanine biosynthesis apart from that seen in tyrB aspC mutant strains. The nucleotide sequences of aspC, ilvE, and tyrB have been determined (111, 192, 193). The coding regions of aspC and tyrB encode 396 and 397 amino acid residues, respectively. These sequences have 169 residues in common, and it seems reasonable to assume that gene duplication played a role in their evolution. The deduced amino acid sequence for the aspartate aminotransferase also agrees with the published amino acid sequence for this enzyme purified from E. coli B (188).
By changing six residues in E. coli AspC to the corresponding residues in E. coli TyrB, i.e., V39L/K41Y/T47I/N69L/T109S and N297S, Onuffer and Kirsch (247) were able to produce mutant enzymes, which had acquired the broadened substrate specificity of the TyrB enzyme. X-ray crystallographic analysis of key inhibitor complexes of this hexamutant revealed that the tyrosine aminotransferase is able to undergo a conformational switch in which an active-site arginine can move aside to allow access of aromatic ligands (217). A mutant form of the TyrB enzyme in which the six substitutions change to the AspC sequence, i.e., S109T/S297N/L39V/Y41K/I47T and L69N shows a nearly ninefold preference for aspartate over phenylalanine (297).
Preliminary crystallographic analysis of the TyrB enzyme shows a dimer in which each monomer binds a molecule of pyridoxal phosphate via a covalent bond linked to the ε-NH2 of Lys258 (185). The gene for the branched-chain aminotransferase, ilvE, encodes a smaller protein of 309 amino acids and does not show homology to either aspC or tyrB (193). The availability of extensive sequence data on aminotransferases from a large number of different organisms has allowed an investigation of their phylogeny. There is an aminotransferase superfamily comprising four families. The TyrB and AspC proteins are closely linked in subfamily Iα and the IlvE aminotransferase is located in the more distant Family III (173).
Using highly purified preparations of the aromatic aminotransferase, chorismate mutase-prephenate dehydrogenase, and chorismate mutase-prephenate dehydratase, Powell and Morrison demonstrated protein-protein interactions between the aromatic aminotransferase and each of the other two enzymes. This interaction did not occur if the aspartate aminotransferase was substituted for the aromatic aminotransferase. The complex with mutase-dehydrogenase dissociated in the presence of tyrosine, and the interaction with mutase-dehydratase required the presence of phenylpyruvate. The interactions therefore are quite specific. The extent to which these interactions occur in vivo and the role they play in synthesis has yet to be determined (269).
As shown in Fig. 3, the first reaction of the terminal pathway of tryptophan biosynthesis involves the conversion of chorismate and glutamine to anthranilate, glutamate, and pyruvate. The enzyme that carries out this reaction is anthranilate synthase (20, 124). Much of the early work has been reviewed elsewhere (378).
The active anthranilate synthase enzyme is an aggregate, an α2β2 heterotetramer, composed of two molecules of each of the polypeptides specified in both E. coli and serovar Typhimurium by the trpE and trpD genes. These are referred to as component I and component II, respectively. The necessity for both components in the reaction was established by studying mutants with nonsense mutations in these genes (31, 169). The aggregate has been purified from both E. coli and serovar Typhimurium. Component I has been purified from E. coli and serovar Typhimurium and component II has been purified from serovar Typhimurium (151, 168, 241). Component I has a molecular weight of about 60,000 and contains the binding site for chorismate. In the absence of component II, component I cannot catalyze the formation of anthranilate by using glutamine as the nitrogen source. It can, however, form anthranilate with a considerably reduced efficiency if provided with ammonia instead of glutamine. Component II also has a molecular weight of about 60,000 and specifies two activities with independent structural domains. The first of these, a glutamidotransferase activity, which is found in the amino-terminal domain, is required to activate component I in the anthranilate synthase reaction. In some microorganisms this function is separately encoded in a gene referred to as trpG (244). This activity channels the nitrogen from glutamine to the active site for anthranilate production. Only the aggregate exhibits this activity, which is stimulated by chorismate. Glutamine hydrolysis requires the prior binding of chorismate to the aggregate (251).
The second activity of component II, present in the carboxyl-terminal domain, converts anthranilate to anthranilate-5-phosphoribosyl pyrophosphate (PRA). It is termed anthranilate phosphoribosyltransferase. This activity can be carried out either by the aggregate or by purified component II. In many microorganisms it is encoded in a gene trpD, part of a cluster of trp genes but not fused with trpG.
When anthranilate synthase from serovar Typhimurium is subjected to limited proteolysis with trypsin, PRA transferase activity is lost and the aggregate is reduced in size from about 261,000 to about 141,000. Analysis of this lower-molecular-weight aggregate shows that component II has been reduced to a polypeptide with a molecular weight of about 15,000 to 19,000. The aggregate with this reduced component II nevertheless retains anthranilate synthase activity, including glutamidotransferase activity (162). Studies of various trpD nonsense mutants reveal that the glutamine-binding site of component II is found in the amino-terminal end of the molecule (373).
In Serratia marcescens, the enzymes glutamidotransferase and PRA transferase are specified by separate genes (trpG and trpD, respectively). The DNA sequences of the end of trpG and the beginning of trpD, including the intercistronic region between them, have been compared with the sequences of the corresponding regions of the bifunctional trpD genes of serovar Typhimurium and E. coli. The intercistronic region is 13 bp long. A hypothetical deletion of one base from this sequence along with a limited number of base substitutions that remove the stop codon of trpG and the ribosome-binding site of trpD, yields a simple scheme in which a gene like the trpD gene of E. coli or serovar Typhimurium could be formed by a gene fusion involving trpG and trpD genes of some ancestral strain (245).
The crystal structures have now been solved for anthranilate synthase aggregates from three different organisms, the archebacterial thermophile Sulfolobus solfataricus (184), S. marcescens (311), and serovar Typhimurium (237). The aggregate for S. solfataricus consists of a TrpE-TrpG heterotetramer, as does the aggregate from S. marcescens. In the case of serovar Typhimurium, the aggregate that is used is a partial complex, a heterotetramer of TrpE and the TrpG component of the TrpD polypeptide. Although there are important quaternary structural differences between the enzyme from S. solfataricus and the other two enzymes, all three show clearly distinct and well separated binding sites for chorismate and the inhibitor tryptophan. This offers confirmation for the hypothesis that tryptophan prevents chorismate binding by conformational effects on the protein. The structures also confirm the role postulated for a number of residues in catalysis or inhibition identified in mutational studies.
A study of a particular trpE mutant TrpEH398M of serovar Typhimurium has revealed that 2-amino-2-deoxychorismate (ADIC) is an enzyme-bound intermediate in the conversion of chorismate to anthranilate, which proceeds in two discrete steps. The activity producing this compound has been termed ADIC synthase. The activity carrying out the next step, the conversion of ADIC to anthranilate, has been termed ADIC lyase. Both activities are present in the TrpE polypeptide and are inhibited by tryptophan (236).
The amino acid sequence of the TrpE component of anthranilate synthase shows close homology with the sequences of two other chorismate-utilizing enzymes, 4-amino-4-deoxychorismate synthase (ADCS), the first enzyme in the synthesis of para-aminobenzoic acid and isochorismate synthase, the first enzyme in the pathway leading to enterochelin, also known as enterobactin (207, 328).
The X-ray structures for two of these proteins, anthranilate synthase and ADCS, have been resolved and reveal marked similarities in structure (184, 237, 254, 311). A single enzymatic mechanism to explain the activities of each of these enzymes has been proposed (147).
Nonsense mutations in trpE can be shown to have a much more dramatic effect on the expression of trpD than on the more distal genes trpC, trpB, and trpA. The basis for this difference was revealed when it was shown that efficient translation of trpD depends on efficient translation of the end of trpE (248). A clue to the molecular basis for this translational coupling was discovered when DNA sequence analysis showed that the translation stop signal of trpE overlaps the translation start signal of trpD in the sequence TGATG (245). The same phenomenon is observed with the translation of the trpB and trpA polypeptides, in which the same overlapping codons are found (267).
As has been found for the first enzyme in a number of biosynthetic pathways, the activity of anthranilate synthase is inhibited by the end product of the pathway, in this case, tryptophan (124, 167). Tryptophan inhibits both anthranilate synthase activity and PRA transferase activity of the aggregate. Whereas anthranilate synthase can be inhibited 100%, PRA transferase inhibition is incomplete, not exceeding 70%. Uncomplexed PRA transferase is not sensitive to inhibition by tryptophan. The binding of tryptophan and chorismate is competitive and both sites are postulated to be present on component I. Mutational studies, however, show that the two binding sites are distinct. Conformational changes involving both component I and component II are associated with the binding of substrates or inhibitor and affect anthranilate synthase, glutamidotransferase, and PRA transferase activities (251).
A number of mutations that affect either the catalytic or feedback-sensitive site of anthranilate synthase of serovar Typhimurium have been identified (46). With these mutated subunits, hybrid complexes that have one catalytically active feedback-insensitive and one catalytically inactive feedback-sensitive subunit with two wild-type PRA transferase subunits have been assembled in vitro. Such a complex is still sensitive to inhibition by tryptophan, indicating the ability of the feedback-sensitive subunit to communicate with and affect the activity of the catalytically active feedback-insensitive subunit present on a separate molecule in the complex (47).
The DNA sequences of the trpE genes of both E. coli and serovar Typhimurium have been compared. They show a high degree of homology at the amino acid level, with only a 12.5% difference in amino acids. The differences at the DNA level are significantly higher but involve a large number of synonymous codon changes. It is interesting that TrpE polypeptides from both organisms do not contain any tryptophan residues (376).
PRA is converted to 1-(O-carboxyphenyl amino)1-deoxyribulose 5-phosphate (CDRP), and this compound is in turn converted to InG phosphate (InGP) by a single enzyme specified by the trpC gene. PRA and CDRP were first postulated as intermediates by Yanofsky (368). CDRP was identified in the dephosphorylated form (CDR) as an accumulation product when certain tryptophan auxotrophs of A. aerogenes and E. coli were starved for tryptophan (100). Using cell extracts of mutants blocked between anthranilate and InGP, Smith and Yanofsky were able to establish that the phosphorylated compound CDRP was an intermediate between anthranilate and InGP (308). In some mutants unable to form InGP, the conversion of CDRP to InGP was blocked, whereas in others, the blocked reaction was the conversion of anthranilate to CDRP. Partial purification of the enzyme from E. coli showed that the reaction CDRP to InGP involved a single enzyme (125). Doy et al. (101) extended the work with cell extracts from auxotrophic strains and established that N-(5'-phosphoribosyl) anthranilic acid was an acid-labile intermediate between anthranilate and CDRP. Because of its extreme lability, the conversion of anthranilate to PRA may have been overlooked in previous investigations. The complete purification of the enzyme PRA isomerase-InGP synthase was achieved in 1966 (76). The enzyme was shown to be a single polypeptide chain with a molecular weight of approximately 45,000, and since the ratio of PRA isomerase activity to InGP synthase activity was the same in the crude cell extract and the purified enzyme, it was concluded that the two activities reside on the same polypeptide. A genetic and biochemical study of missense mutations in the trpC gene established a relationship between the map position of the mutation and the enzymatic function lost. Strains with mutations in the proximal half of the trpC gene (closest to trpD) accumulated CDR; i.e., they lost InGP synthase activity. On the other hand, when the mutation lay in the distal region of trpC (closest to trpB), anthranilic acid accumulated, indicating a loss of PRA isomerase activity (307). Limited proteolysis of the purified enzyme has allowed the isolation of large amino-terminal and carboxy-terminal fragments of the enzyme, which retain InGP synthase and PRA isomerase activities, respectively (181). In some other microorganisms the gene for PRA isomerase (trpF) and InGP synthase (trpC) are separate (244).
The three-dimensional structure of PRA isomerase-InGP synthase from E. coli has been determined to 2.0 Å resolution (350). The InGP synthase activity is located in the amino-terminal domain of the protein and the PRA isomerase in the carboxyl-terminal domain. Both of these domains possess structurally identical βα (7-8) subdomains for binding the phosphate moieties of the relevant substrates and products and it has been proposed that they may have evolved from a common ancestor (317, 350). These proteins along with the α−subunit of tryptophan synthase form a subfamily involving the triose phosphate isomerase (TIM) (24). The role of conserved residues in the active site of InGP synthase of E. coli has been confirmed by extensive mutagenesis (83).
The trpC gene in E. coli is flanked by intercistronic regions of 11 nucleotides (trpC-trpB) and 6 nucleotides (trpD-trpC) and in this way differs from the trpE-trpD and trpB-trpA junctions (60).
Tryptophan synthase was one of the first enzymes of the tryptophan pathway to be extensively studied and has also been the subject of a number of review articles (226, 227, 369, 370). The enzyme carries out three reactions directly relevant to tryptophan biosynthesis (226, 227, 366, 375).
InGP → indole + d-glyceraldehyde 3-phosphate (i)
Indole + l-serine → l-tryptophan + H2O (ii)
InGP + l-serine → l-tryptophan + d-glyceraldehyde 3-phosphate + H2O (iii)
A consideration of the rates of the three reactions pointed to reaction iii as the major physiological reaction. This was supported by the demonstration that free indole was not an intermediate in the reaction (74, 75, 368, 375).
Tryptophan synthase is a tetramer of two α-units specified by the trpA gene and a β2-dimer specified by the trpB gene. The β2-subunit has two binding sites for pyridoxal phosphate and two independent sites to which α-units can bind (74, 375). The α-subunit has the ability to convert InGP to indole and glyceraldehyde 3-phosphate and the β2-subunit is able to convert indole and serine in the presence of pyridoxal phosphate to tryptophan. In both cases, however, the α2β2 complex carries out these reactions at greatly increased rates.
The α-unit has been purified and crystallized and has a molecular weight of 29,000 (152, 181, 295). The amino acid sequence of the α-subunit has been determined and, like the trpE-encoded enzyme, it does not contain any tryptophan residues (206, 370). The analysis of the amino acid substitutions that occurred in certain trpA mutants and the map location of the mutations causing these changes provided the first compelling evidence of colinearity between the gene and the polypeptide encoded by it (371).
The β2-subunit has also been purified and crystallized. It has a molecular weight of 89,000 (1, 2, 127, 144, 145, 371). The native α2β2-complex (molecular weight, 147,000) has been purified and crystallized from E. coli and shown to be identical with the reconstituted tryptophan synthase complex (1, 138). The tryptophan synthase complex has also been crystallized from serovar Typhimurium (4). Resolution of this structure to 2.5 Å has allowed the formulation of a detailed three-dimensional structure (163). An essential feature of this structure is a hydrophobic tunnel of about 25 Å linking the active sites of the α− and β-subunits. The diameter of this tunnel matches that of the intermediate substrate indole and it has been proposed that this tunnel facilitates the movement of indole from the active site of the α-subunit to the active site of the β-subunit. It has also been proposed that the tunnel prevents the escape of indole to the solvent during catalysis (5, 43, 105, 163, 364). Further structural studies involving wild-type and mutant forms of the tryptophan synthase α2β2-complex with different ligands bound to the active sites have revealed ligand-induced conformational changes postulated to be important for the channeling of indole and for allosteric communication between the α− and β-subunits (273, 274, 275, 292). A detailed review of the various molecular interactions associated with the tunnel and the transfer of indole has been published (228). Several kinetic and structural studies have established the mechanisms by which allosteric interactions involving both the α− and β-subunits synchronize the reactions taking place at both ends of the tunnel (9, 43, 292). The reaction of serine with the cofactor pyridoxal phosphate at the β-site stimulates the formation of indole at the α site and its passage through the tunnel ensuring that indole does not reach the β-site before the highly reactive enzyme-aminoacrylate, has been formed. Because of the fast and irreversible reaction of indole with the aminoacrylate, this prevents the accumulation of indole in the tunnel. It has also been shown that binding of various ligands to the α-site induce extensive conformational changes in the α2β2-complex which prevent the escape of indole to the solvent and favor the formation of E-aminoacrylate at the β site (3, 43, 243, 252, 319).
The capacity of a microorganism to efficiently utilize amino acids, which may be present in the environment, is as important as many of the controls regulating endogenous biosynthesis. There are at least three transport systems that efficiently take up phenylalanine or tyrosine from the environment. Each of these systems involves a polytopic integral membrane protein whose activity is energized by the proton motive force. The first to be identified in both serovar Typhimurium and E. coli was the so-called general aromatic transport system, which transports each of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan with high affinity (Km = 0.4 μM) and is encoded by the aroP gene (7, 8, 40, 61). The AroP protein is a protein of 457 amino acids and has been shown to contain 12 transmembrane spans with the amino-terminal and carboxy-terminal regions in the cytoplasm (72). The transcription of the gene encoding this protein is regulated by the TyrR protein, which represses expression in the presence of either phenylalanine, tyrosine, or tryptophan (346). The complex mechanism by which this is achieved is discussed in more detail later. The AroP protein is a member of the amino acid transport (AAT) family within the superfamily of amino acid-polyamine-organocation transporters (377).
A second protein encoded by a gene pheP also transports phenylalanine (344). The PheP and AroP proteins are closely related, sharing 61% identical residues. However, PheP, which is principally a transporter of phenylalanine with a Km of 2 μM, is expressed at low levels in the cell. When the level of expression is increased by changing the translational start codon from GTG to ATG or by increasing copy number, the protein now exhibits significant transport of tyrosine even though the Km for tyrosine is significantly higher (30 μM) than that for phenylalanine (71). The basis for this change is not understood but the low level of PheP expression in wild-type cells may well favor specificity for a single amino acid, i.e., phenylalanine. Extensive studies have been carried out identifying critically important residues in the protein, establishing topology and some aspects of a putative tertiary structure (71, 97, 256, 257, 258, 259, 260, 382). The LIV-I/LS branched-chain amino acid transporter can also transport phenylalanine, with Km values in the LIV-I and LS systems being 19 and 30 μM (189). It remains to be seen whether this capacity explains the earlier reports of a locus azaB, mutations of which impaired active transport of phenylalanine in aroP mutants (349).
The tyrP gene encodes a protein, which specifically transports tyrosine. Along with two tryptophan-specific transporters, Mtr and Tna, TyrP belongs in a small family of transporters (HAAP), which appear to have 11 transmembrane spans with the carboxy terminus in the periplasm (286, 353). The expression of the tyrP gene is also regulated by the TyrR protein, which, in the presence of tyrosine, represses expression and, in the presence of phenylalanine or tryptophan, causes enhanced expression (12, 346).
Two tryptophan-specific transport system involve the TnaB and the Mtr proteins. The TnaB system, which is characterized by high capacity, but low affinity, is induced when tryptophan is being used as a sole carbon source. The transport protein has a Km for tryptophan of 10 μM and is synthesized along with the enzyme tryptophanase, which is encoded in the same transcription unit. The mtr gene encodes a high-affinity tryptophan transport system, whose expression is repressed by the TrpR protein in the presence of tryptophan. In the absence of tryptophan, expression of mtr is activated by the TyrR protein with either phenylalanine or tyrosine (150, 287, 288). This transporter, which has a Km for tryptophan of 2 μM, plays a major role in the uptake of tryptophan for protein synthesis.
In addition to the transporters of the aromatic amino acids, E. coli has another integral membrane protein ShiA, which transports the common pathway intermediate shikimic acid (343). This protein shows significant homologies with a number of proteins in cluster 3 of the major facilitator superfamily (219), but its overall role in the physiology of the cell is not clear.
The chromosomal locations of the structural genes for the enzymes of the pathway of biosynthesis of phenylalanine, tyrosine, and tryptophan, their transport systems, and the pathway's two major regulator genes in E. coli are shown in Fig. 4. Full details of genes and enzymes are now available from a number of electronic sources such as EcoCyc at http://EcoCyc.org/ or the E. coli Genetic Stock Center (CGSC) database at http://cgsc.biology.yale.edu.
The flow of substrates along the pathway is controlled by feedback inhibition of each of the DAHP synthase enzymes and the three major enzymes utilizing chorismate, i.e., anthranilate synthase, chorismate mutase-prephenate dehydrogenase, and chorismate mutase-prephenate dehydratase. These interactions have been discussed in previous sections.
Expression of genes of the trp operon and also some of the common pathway genes is modulated by a repressor-independent system termed metabolic regulation by Rose and Yanofsky (280, 323). The synthesis of a number of key enzymes and transporters is regulated in response to fluctuations in the intracellular concentration of the three aromatic amino acids and involving one or both of the regulatory proteins, TyrR and the TrpR. In addition, there is another system of transcriptional control called attenuation, which modulates gene expression in response to the availability of specific charged aminoacyl-tRNAs. The trp operon and the pheA gene are both subject to transcriptional control by attenuation. We will consider these specific controls separately under the headings, "TyrR protein and the TyrR regulon," "TrpR protein and the TrpR regulon," and "Attenuation."
The TyrR regulon comprises at least nine separate transcription units (41, 42, 48, 49, 92, 107, 130, 150, 164, 165, 221, 239, 253, 287, 334, 346, 362, 364). TyrR protein can act as a repressor and, in some cases, as an activator of transcription. The effector molecules ATP, tyrosine, phenylalanine, and, to a lesser extent, tryptophan are able to affect its biological activity. This system has been reviewed (262, 263).
The tyrR gene has been cloned, and its sequence has been determined (69, 70, 358). From the sequence, the TyrR protein comprises 513 amino acids with a calculated subunit molecular weight of 57,640, compared with a measured molecular weight of 110,000 + 5,000 for the homodimer, in which form the protein normally occurs in the absence of effectors (15, 80).
The protein has been overexpressed and purified in a number of laboratories (11, 15, 80). Limited proteolysis indicates that the protein has three functional domains. The amino-terminal domain involves residues 1 through 190, the central domain residues 191 through 467, and the third domain residues 468 through 513 (80) (see Fig. 5). Each of the domains is important for a different function of the TyrR protein. They have all been independently expressed and the amino-terminal domain has been crystallized (96, 194, 215, 325a).
The amino-terminal domain.
Deletions and specific amino acid substitutions have identified the region of the protein between amino acids 2 and 19 as playing a critical role in activation of the expression of the genes tyrP and mtr. A number of mutations affecting this region abolish TyrR-mediated activation without affecting repression of the other members of the regulon (78, 79, 357, 359). Alanine scanning mutagenesis has identified residues 9 and 10 as critical for activation and also identified a second critically important region involving D103 (355). A glycine at position 37 also appears to play an important role. Iterative database searches have identified a ligand-binding domain (ACT) based on the C-terminal domain of 3-phosphoglycerate dehydrogenase. In the case of TyrR, this ACT domain occupies position 2-72 of the amino-terminal domain (14, 110). A second domain referred to as PAS (110) and implicated in signal transduction has been identified in the region between residues 80 and 114. Verger et al. (325a) have determined the amino-terminal domain of TyrR to a resolution of 2.3 Å. In agreement with previous genetic and bioinformatics data, the amino-terminal domain was found to contain a double βαβ ATC domain (residues 1 to 68) and an α+β PAS domain (residues 89 to 175) separated by a connecting helix (residues 76 to 88). The last 13 amino acids (residues 178 to 190) form a final helix.
In vitro transcription studies have established that activation of both the tyrP and mtr genes requires an interaction between the TyrR protein and the carboxy-terminal region of the α-subunit of RNA polymerase, as has been shown for other class I activators (166, 199, 357). Substitutions DN250 and RE310 in the α-subunit of RNA polymerase destroy TyrR-mediated activation without affecting the interaction of the mutant alpha with UP sequences and may constitute a TyrR-specific patch (362; Camakaris and Fujii, unpublished results). In vitro studies with supercoiled templates of either mtr or tyrP have shown that TyrR-phenylalanine-mediated activation can reverse transcriptional inhibition caused by small DNA-binding proteins such as HU or IHF (355).
The failure to find any mutations specifically affecting activation and not repression in any region other than that coding for the amino-terminal domain, coupled with the demonstration that a mutant deleted for much of the central domain (residues 226 through 419) shows wild-type levels of activation for tyrP and mtr (263), suggests that both a binding pocket for the aromatic amino acids and a region of the protein able to interact with the α subunit of RNA polymerase will be found in the amino-terminal domain. Studies with the purified protein have identified ATP-independent phenylalanine binding, in addition to the ATP-dependent tyrosine binding previously associated with the central domain (351). Structure studies confirm the suggested role of the ACT domain in binding the aromatic amino acids (325a). It seems likely that both the amino-terminal and the central domain have binding sites that recognize the three aromatic amino acids with different affinities. When overexpressed, the amino-terminal domain exists as a dimer indicating that it also contains a dimerization motif (194). Interactions between the ACT domains, the connecting helices, and the final helices of two monomers appear to stabilize dimer formation (325a).
The central domain.
The central domain of TyrR is homologous to the central domains of a number of activator proteins, including NtrC, NifA, DctD, XylR, and FhlA, each of which activate expression from σ54 promoters (discussed in reference 262). Mutational studies of the NtrC protein showed that the central domain of this protein is essential for activation (18, 339). A key component of this central domain is an adenylate-type ATP-binding site. Mutations that cause amino acid substitutions in site A of the ATP-binding site and a number of other mutations in this central domain destroy the ability of NtrC protein to activate gene expression. Furthermore, in the case of NtrC, ATP hydrolysis is involved in converting the RNA polymerase-promoter complex from a closed to an open state. The situation with regard to TyrR is quite different. Mutations altering site A of the ATP-binding site and three other mutations affecting the highly conserved residues E-274, G-285, and E-302 of the central domain have no effect on activation of tyrP and mtr but abolish TyrR-tyrosine-mediated repression of aroF, aroL, tyrP, and tyrB (357). It has been demonstrated (15) that purified TyrR protein binds ATP with a Kd of 5 to 7 μM. In the presence of excess ATP, TyrR protein binds tyrosine with a Kd of 50 μM. Tyrosine binding cannot be detected in the absence of ATP. It has also been shown that, in the presence of the nonhydrolyzable ATP analogue, ATP-γS and tyrosine (500 μM) or phenylalanine (25 mM), TyrR protein self-associates to form a hexamer (352). This hexameric form is postulated to play a major role in tyrosine-mediated repression and in the activation of folA. In the central domain mutant EQ274, both tyrosine binding and tyrosine-dependent hexamerization are significantly impaired, although binding of ATP is unaltered (195). The role of ATP hydrolysis in repression has not yet been resolved. The purified TyrR protein does have ATPase activity that is stimulated by tyrosine (80, 352). Cui et al. (77) estimate that the level of this activity is similar to that reported for the unphosphorylated form of the NtrC protein. Weiss et al. (339) reported that activation of NtrC by phosphorylation increases its ATPase activity about 11-fold. Although mutants of TyrR with amino acid substitutions in site A of the ATP-binding site have been isolated and shown to have lost the ability to carry out TyrR-tyrosine-mediated repression (359), these mutant proteins have not yet been purified and tested for ATP binding and hydrolysis. The finding by Wilson et al. (352) that the nonhydrolyzable ATP analogue ATP-γS can substitute for ATP in the self-association of TyrR dimers into hexamers might indicate that only binding is required for repression. If this is the case, perhaps ATP hydrolysis functions to facilitate the reverse reaction of hexamer to dimers, hence freeing operator sites. In the case of NtrC protein, Austin and Dixon (17) have reported that ATPase activity is stimulated by DNA, which contains NtrC-specific binding sites. In the case of XylR, oligomerization caused by the binding of ATP is necessary both for the activation of the σ54 promoter Ps and for the repression of its own σ70 promoter Pr (35). The TyrR protein has also been shown to have phosphatase activity that is stimulated by Zn2+ and inhibited by tyrosine and its analogues and by ATP and its analogues. The phosphatase active site has been localized within the central domain but its role in TyrR-mediated activities has not yet been elucidated (383).
When the central domain fragment 188-467 is expressed, it forms a monomer. Tyrosine, in the presence of ATP-γS, promotes its oligomerization to a hexamer (96), confirming that the ATP-dependent binding site for tyrosine, the ATP-binding site, and the hexamerization motif are all located in the central domain.
When the amino acid sequence of the central region of TyrR is compared with those of other members of the NtrC family, it can be seen that a small sequence of seven amino acids (EKGAFTG) is missing within the C3 region immediately after residue 279 (238). This deletion is not present in PhhR, a homologue of TyrR present in Pseudomonas aeruginosa and a transcriptional activator of σ54 promoters (310). Mutational analysis of the C3 region of NifA has established that although such mutants are unable to activate expression, they still bind DNA and are able to form oligomers leading to the hypothesis that the GAFTGA subregion may be involved in recognition of the σ54 promoter complex (131). On the other hand, mutants in the same region of the enhancer-binding protein DctD have been shown to retain the ability to cross-link to σ54 and the α-subunit of RNA polymerase and to hydrolyze ATP. It has been proposed that the role for the C3 region may be the coupling of ATP hydrolysis and promoter open complex formation (338).
The carboxyl domain.
The carboxyl domain contains a classic Cro-like HTH DNA-binding motif. Alanine scanning experiments have identified a number of residues in both helix 1 and helix 2 that are essential for DNA binding. The mutant analyses combined with consideration of the known interactions of Cro and CAP have led to the suggestion that R484 and H494 both form critical bonds with the invariant bases (G·C)(C·G)8 (161). Deletion of 10 of the 11 amino acids between helix 2 and the carboxyl terminus also destroys activation and repression functions (359). Similarly, a truncated TyrR protein missing the HTH motif can neither repress nor activate members of the regulon (78). An alanine-substituted mutant TyrR protein HA494 is completely defective in binding to the tyrP operator, whereas another substituted mutant protein TA495 shows reduced binding. Missing contact probing by using this mutant protein suggests that T495 makes specific contacts with adenine and thymine bases at +/− 5 positions in the TyrR boxes. This analysis also reveals the importance for binding of the AT-rich sequences between the palindromic arms of strong but not the weak TyrR boxes (160) (see the next section).
When overexpressed, the carboxy-terminal domain is also shown to exist as a dimer (194). Representation of the TyrR protein showing the three domains and the critical amino acids that have been discussed in this section is given in Fig. 5, and, a discussion of the DNA sites to which the protein binds is included below.
The TyrR boxes and their arrangement.
The target sites on the DNA that are recognized by the 'I'yrR protein are all related to the palindromic sequence TGTAAAN6TTTACA. The bases in boldface type (G and C) are invariant and, changing them to any other base largely destroys the function of the box (10, 62, 65, 113, 161, 177, 200, 287, 288, 354). These palindromic sequences, together with the two flanking nucleotides at each end, are referred to as TyrR boxes. Their distribution with regard to the putative RNA polymerase-binding sites for each of the transcription units is shown in Fig. 6 along with relevant TrpR-binding sites for aroL and mtr. The sequence of each box is shown in Fig. 7. All of the transcription units repressed by tyrosine, i.e., aroF-tyrA, aroL, aroP, tyrB, and tyrP, have two adjacent TyrR boxes separated by a single base. In every case, the box showing the closest agreement to the consensus sequence is bound by the TyrR protein in the absence of effectors (11, 12, 200, 262, 288, 354, 358). For convenience, these boxes are referred to as strong boxes. Strong boxes, with the exception of aroG, are also AT rich in the central six positions (Fig. 7). The adjacent weak boxes are bound by TyrR protein only in the presence of effectors ATP and tyrosine, which in some cases can be substituted by phenylalanine, and only if there is a strong TyrR box nearby on the same face of the helix. In the case of tyrP, this cooperative binding between strong and weak boxes still occurs if the boxes are separated by one turn of the helix, but not if the separation involves three turns of the helix (11). In general, weak boxes have a weaker agreement with the consensus and contain two or more Gs and Cs within the central six bases. In the case of aroF, tyrP, aroL, and tyrB, the weak box is closest to or overlapping the RNA polymerase-binding site.
It has been proposed that the hexameric form of the protein formed in the presence of tyrosine and ATP is able to bind to two or more boxes (352). In some other cases, where there are two boxes, such binding involves two dimers and can be facilitated by the presence of phenylalanine and ATP (358). One exception to the general rule that the weak box plays a major role in tyrosine-mediated repression is the finding that box 2 of aroL apparently plays no part in repression. Like the other weak boxes, it is bound by TyrR protein only in the presence of tyrosine and ATP, but mutating the invariant G-C pairs in this box has no effect on repression, which appears to involve a TyrR-mediated loop between strong boxes 1 and 3, which are separated by 54 bp (200). The presence of two strong boxes separated by three turns of the helix is also found in the case of the aroF promoter, where DNA looping between the strong boxes is again a strong possibility.
Diverse strategies within the TyrR regulon.
A major advantage of the regulon structure is that each transcription unit is unique and has been able to evolve transcriptional controls best suited to the physiological role of the proteins encoded by that transcription unit. In the case of the TyrR regulon these various responses depend on subtle differences in TyrR box composition, their number and location, and, in some cases, the additional action of other transcription factors. These different strategies will be illustrated by a more detailed consideration of the regulation of some of the genes of the regulon.
The TyrB protein is an aminotransferase essential for the biosynthesis of tyrosine and important for the biosynthesis of phenylalanine. In minimal medium the tyrB gene is derepressed but when tyrosine or phenylalanine is added to the medium expression is repressed fourfold and threefold, respectively. As shown in Fig. 6, tyrB has two TyrR boxes located downstream of the promoter. In the absence of aromatic amino acids, TyrR protein selectively binds to the strong box. In the presence of either tyrosine or phenylalanine this binding is extended to include the weak box. When TyrR is bound to both these boxes, RNA polymerase can still bind to the tyrB promoter. However, in the presence of tyrosine, when it is presumed that a hexamer binds across both boxes, RNA polymerase is unable to proceed to the formation of an open complex. In the presence of phenylalanine, when it is presumed that two dimers bind cooperatively to the two boxes, open complexes can be formed, but the RNA polymerase is prevented from leaving the promoter and actively initiating transcription. By inserting bases between the TyrR boxes and the promoter it could be shown that both of these effects disappeared if the boxes were moved more than 19 bases away. So the dependence of TyrR on tyrosine or phenylalanine, in order to bind the weak box, and the positioning of that box close to the transcription initiation site ensures effective transcriptional control by tyrosine and phenylalanine. The fact that the TyrR boxes are located near the transcription start site rather than overlapping the –35 region of the promoter also ensures that the aromatic amino acids will not cause activation of tyrB expression (358).
The gene aroP also has two TyrR boxes located downstream of the promoter (Fig. 6). However, in this case, the arrangement of strong and weak boxes is the reverse of what would be expected, with the strong box closest to the promoter. Furthermore, both boxes are separated from the promoter by a distance, which, in the case of tyrB, has been shown to nullify repression. The resolution of this dilemma came with the discovery of a promoter (P3; see Fig. 6) on the opposite strand which was nonproductive but which could nevertheless bind RNA polymerase when TyrR protein bound the TyrR boxes in the presence of one or other of the aromatic amino acid effectors. When an RNA polymerase molecule binds to this promoter, RNA polymerase is no longer able to occupy either of the promoters (P1 or P2) that would lead to transcription of the aroP gene. This effect depended not on TyrR protein’s ability to repress but on its activation functions, as demonstrated by the fact that activation-minus mutants of TyrR (RQ10) could not repress aroP, whereas the mutant TyrR EQ274, which is impaired in other cases of tyrosine-TyrR-mediated repression, is able to repress aroP. The AroP protein is a transporter that actively transports each of the three aromatic amino acids into the cell. The system that has evolved to control its synthesis utilizes the activation functions of TyrR, which can respond to each of the three aromatic amino acids (335, 336, 337).
The gene aroL encodes an important enzyme in the common pathway of aromatic amino acid biosynthesis and its expression is repressed by TyrR-tyrosine (fivefold) with significant but weaker repression by TyrR-phenylalanine (twofold). The addition of tryptophan further increases repression (93). In this case, the tryptophan effect depends on the TrpR protein and the presence of a TrpR-binding site immediately downstream of the TyrR boxes (149, 360). The TrpR-tryptophan effect is only observed in tyrR+ strains and disappears if the TrpR and TyrR boxes are moved apart by half a turn of the helix suggesting that interactions between these two proteins may play a role in the tryptophan effect (200). Furthermore, mutational studies show that in this case, although there is a weak TyrR box overlapping the transcription start site, repression depends on the two strong boxes lying at either end of the promoter region and probably involves TyrR-mediated looping of the DNA (200).
Another gene whose expression is repressed by tryptophan and involving the TrpR protein is the gene for the tryptophan-specific transporter mtr. This gene is also subject to TyrR-mediated control, but, in this case, the TrpR effect is clearly dominant (150, 287, 288). As can be seen in Fig. 6, the TrpR-binding site overlaps the promoter while the two TyrR boxes are located upstream of the –35 sequence. In the absence of tryptophan and in the presence of either phenylalanine or tyrosine, TyrR protein bound to the TyrR boxes can activate expression of the mtr gene. When tryptophan is missing from the aromatic amino acid pool, the cell increases its efforts to capture any tryptophan in the environment.
The gene tyrP, which encodes a tyrosine-specific transporter, has evolved a similar control system, in that it is fully repressed in the presence of tyrosine but activated to higher levels in the absence of tyrosine and the presence of either phenylalanine or tryptophan. This is achieved by positioning adjacent strong/weak TyrR boxes so that the weak box overlaps the –35 sequence (Fig. 6). In the presence of tyrosine, the TyrR hexamer binds to both boxes and prevents the RNA polymerase from binding to the tyrP promoter (361). Both TyrR protein and α-subunit of RNA polymerase compete for binding to critical bases in the region of the weak box (52, 160). In the presence of phenylalanine, and to a lesser extent tryptophan, TyrR protein dimers bind to the strong box and interact with the α-subunit of RNA polymerase to increase binding and enhance open complex formation (199, 356). Phenylalanine can also facilitate the cooperative binding of two TyrR dimers to a strong/weak box combination and if the level of TyrR protein is increased, or an activation-defective mutant is used, phenylalanine like tyrosine represses tyrP expression. These results indicate that the intracellular levels of TyrR protein are delicately balanced to achieve particular regulatory outcomes. The strong TyrR box is not optimally placed for activation, which is improved by moving it 3 or 4 bases upstream, but its position is optimal with regard to the placement of the adjacent weak box and effective repression, which would appear to be the dominant response (11).
Three other transcription units show a very significant response to changes in the level of TyrR protein. These are the aroF-tyrA operon and aroG, each of which encodes a gene for one of the DAHP synthase isoenzymes, and tyrR, the gene for the TyrR protein. Regulation of aroG and tyrR appears to be unaffected by the presence or absence of aromatic amino acids, although early studies in a chemostat had suggested a fourfold derepression of aroG in cultures of an aromatic auxotroph starved for phenylalanine or tryptophan. On the other hand, growth of wild-type cells in the presence of all aromatic amino acids, caused only a 20% drop in activity, and similar small changes were observed in studying the expression of an aroG-lac construct (27, 28, 42, 48, 85, 164). Changing the levels of TyrR protein either by introducing additional copies of the gene on a multicopy plasmid or by mutating the tyrR gene has a marked and unambiguous effect on expression from the aroG and tyrR promoters. The aroG gene has a single strong TyrR box in a position identical to that of the weak box in tyrP (Fig. 6). It is an unusual box in that, although the palindromic arms exactly fit the consensus, the sequence between them is GC rich (Fig. 7). Nevertheless, it is bound by TyrR protein in the presence of ATP without any phenylalanine or tyrosine (28). In the light of the results with tyrP, it would therefore seem that repression of aroG is a consequence of direct competition between TyrR protein and RNA polymerase binding to the DNA. The situation with tyrR is less clear, because the strong TyrR box and a second weak box are located upstream of the –35 hexamer (Fig. 6) (16). No studies to investigate the binding of both TyrR protein and RNA polymerase to this promoter have yet been carried out.
The promoter of the aroF-tyrA operon shows about a 10-fold repression in minimal medium, which is due to normal levels of the TyrR protein alone. When tyrosine is added, expression is repressed a further 10- to 20-fold. If levels of TyrR protein are increased, phenylalanine can also cause repression. As can be seen in Fig. 7, the aroF promoter has three TyrR boxes. As is the case with tyrP, separating boxes 1 and 2 by half a turn of the helix completely destroys repression (11). On the other hand, inactivating box 3 or moving it up to 110 bases upstream only partially reduces repression (64, 65). Box 2 is a very AT-rich sequence and its position corresponds to that of known UP sequences that are recognized by the α-subunit of RNA polymerase and can greatly enhance promoter activity (133). It is possible that even in the absence of the aromatic amino acids, by binding to strong box 2, TyrR protein can partially interfere with the interaction between RNA polymerase and the extended promoter. Whatever the mechanism, it is of particular interest that both the aroF and aroG promoters have reserve strengths that are only realized when levels of TyrR protein in the cell are diminished. These two enzymes compete with other enzyme systems for the substrates PEP and E4P and under certain physiological circumstances may need to be synthesized in greatly increased amounts. For example, when tyrR+ strains of E. coli are grown in minimal medium supplemented with all amino acids except the three aromatic amino acids the expression of aroF is fully derepressed well above minimal medium levels to those observed in tyrR mutant strains (322). The complementary studies on the levels of TyrR protein under these conditions have not yet been carried out.
The application of the Genomic Selex method as described by Shimada et al. (299), using purified TyrR protein and randomized fragments of the E. coli chromosome, has been used to search for additional genes whose expression may be regulated by TyrR protein (362). This identified a single new member of the regulon, namely folA. Expression of folA is activated by TyrR protein in the presence of tyrosine and IHF. The distance between the two TyrR boxes and the −35 region of the promoter is significantly greater than the distance that will allow activation of tyrP and, unlike activation of tyrP, which does not require a fully functional central domain, activation of folA requires all three domains of TyrR to be fully functional. It would appear that the combined effects of hexamerization and IHF are required to position the TyrR protein close enough to the promoter to activate RNA polymerase. There are many similarities between this system and the reported activation of tpl by TyrR-tyrosine in Citrobacter (19, 305). Although folA was the only gene in addition to the known members of the TyrR regulon for which TyrR-regulated expression could be demonstrated, there were some other genes with TyrR-binding sites located within or close to promoter sequences and more work needs to be done before totally excluding TyrR-mediated regulation of these genes.
The trpR gene was first described in 1959 (67). The TrpR protein was one of the first repressor proteins to be identified in E. coli and its study contributed significantly to the elaboration of the Operon model as applied to repressible systems. The trpR gene was cloned independently by Gunsalus et al. (141), and by Roeder and Somerville (277), and its sequence was determined. From the sequence, the translation product of the gene was deduced to be a polypeptide of 108 amino acids. The protein was shown to exist as a dimer (175). A mutational analysis carried out by Kelley and Yanofsky (179) to look for negative dominant mutations identified a putative helix-turn-helix motif that contained amino acids essential for repressor action and that was located toward the carboxyl end of the protein. The protein was crystallized and its structure was determined to a resolution of 2.6 Å (289) and subsequently to 1.8 Å (379). It is mainly a helical structure. Each monomer comprises six helices (A through F), and the dimeric protein has a structure in which five of the six helices are interlinked. Two molecules of tryptophan are bound per dimer and the binding of tryptophan alters the critical orientation of the HTH substructure essential for operator-specific recognition (220). When tryptophan binds to the aporepressor, the two E helices of the dimer move 5 to 9 Å apart so that they fit into adjacent major grooves of the DNA (209, 379). A review by Somerville details many of the studies involving the TrpR protein up until 1992 (309). Although its crystal structure was determined relatively early in these studies, the exact mode of interaction between the TrpR protein and its various DNA targets has been the subject of many varied and sometimes conflicting studies.
The TrpR regulon comprises five transcription units, namely, the trpEDCBA operon (30, 33, 183, 191), aroH (41, 137), trpR (37, 140, 180), mtr (148, 287), and aroL (149, 200). The trpEDCBA operon has been extensively studied and along with aroH and trpR is regulated by TrpR protein in the presence of tryptophan. The TyrR repressor has no effect on the expression of the trp operon or trpR. A possible effect on aroH is yet to be confirmed. As described earlier, TyrR is involved in the regulation of mtr and aroL. Early studies of the crystals of the Trp repressor bound to a 19-bp DNA target based on the putative binding site in front of the trpEDCBA operon produced the surprising conclusion that, with one exception, there were no direct interactions between amino acids in the binding head of the protein and the nucleotides in the operator, but that there were a number of hydrogen bond interactions mediated by water molecules (174, 202, 250, 289). Other studies showed that the protein could also recognize the shape of the DNA sequence (25, 171, 250). Finally, a second crystal structure involving the protein complexed to a longer sequence has shown that two dimers can bind cooperatively with protein-protein interactions contributing significantly to stability (201). The involvement of amino acids in the amino-terminal arms of the repressor in this interaction has also been demonstrated (212, 298).
The operator sequence for the trpEDCBA operon was first identified by the isolation and characterization of various operator constitutive mutants (33). The point mutants that were isolated had base pair changes at positions −6 and −5 and +5 and +6 (Fig. 8). In a separate study that used the challenge phage system, Bass et al. (29, 30) made symmetrical base substitutions at 11 positions in the trp operator. Mutations affecting CTAG at positions −6 −5 −4 −3 and +3 +4 +5 +6 revealed the importance of this sequence in operator function. Studying an alternate sequence based on the right arm of the trp operator with the palindromic CTAG as the center of symmetry, Staacke et al. (315) also confirmed the importance of the sequence ACTAGT in TrpR binding. In a separate study, Czernik et al. (81) used a TrpR protein affinity column to select from a randomized pool of synthetic double-stranded DNA those sequences specifically bound by the column in the presence of tryptophan and eluted on the addition of the tryptophan analogue β-indole acrylic acid. Analysis of these sequences also identified CTAG as the motif that was critical for binding. Notwithstanding these results, examination of the sequences in the vicinity of the promoters for each of the five transcription units of the TrpR regulon failed to identify an unambiguous and conserved binding site present with each member of the regulon. After considerable debate and more experimentation, it now appears that the sequence recognized by the DNA-binding domain of the repressor is GNACT (136, 172, 360). When two such sequences are separated by 8 bp, a dimer can bind to two successive major grooves. When GNACT sequences are separated by only three bases two dimers can bind next to each other, gaining stability from protein-protein interactions and sharing the same major groove as the helix approaches the DNA end on (139, 201). Under these circumstances the additional two half-sites, which bind the second binding domain of each dimer, do not have to maintain a close agreement with the consensus.
The explanation for the critical role of CTAG depends on its palindromic nature and the demonstration of the tandem binding of juxtaposed dimers in which a CTAG half-site is shared by monomers from two different dimers (201). This model is particularly relevant to the aroL and mtr binding sites (172).
Examination of the nucleotide sequences of the various promoters of the members of the TrpR regulon, combined with structural studies, in vitro and in vivo protection experiments have revealed the distinctive way in which TrpR protein binds to each of these sequences. Figure 9, which is adapted from the paper by Yang et al. (360), provides a credible model for these complex interactions, in which sequence recognition and interactions between dimers play an important part. Their results are in agreement with those of Kumamoto et al. (191), who concluded that, whereas one dimer bound to the trpR operator, two bound to aroH and three to trp. There is, however, still no complete agreement on the number of dimers that bind in each case, as the gel retardation and DNase I protection studies reported by Jeeves et al. (172) show that, under their conditions, the tandem binding of two TrpR dimers occurs with the trpR operator and a 3:1 complex is observed with the aroH operator. This paper also provides the experimental validation for the proposed binding of TrpR to the mtr and aroL TrpR-binding sites.
The level of repression, that is, the ratio of fully derepressed to fully repressed levels, is different for each of these units. It is about 100-fold for the trp operon, 6-fold for aroH, and 3-fold for trpR. The degree of repression of the aroH-specific promoter may be significantly greater than sixfold because Hudson et al. (158) have reported significant readthrough into aroH from an upstream URF whose expression is not regulated by TrpR. Muday et al. (239) have reported that the tyrR+ allele has an enhancing effect on TrpR-mediated repression of aroH. They also identified a putative weak TyrR box 38 bp downstream of the TrpR-binding site. No mutational or binding studies have been carried out to confirm the role of this putative binding site in aroH regulation and unlike aroL the putative TyrR box is located a significant distance apart from the TrpR-binding site. However, because of the synergy between these two proteins that is evident in the regulation of aroL, further studies should be undertaken to confirm this preliminary result and to establish whether aroH should also be included in the TyrR regulon.
The mtr gene along with arol is part of the TyrR regulon. The repression ratio for mtr is about 100, whereas in the case of aroL, TrpR-trp only strengthens the repression brought about by TyrR-tyr but has no effect in strains lacking functional TyrR protein. There are probably complex reasons for these significant differences in repression ratios that involve major differences in promoter strengths, the number of dimers binding to each operator, the placement of the binding sites relative to key regions of each promoter, and possible interactions with other regulatory protein.
Regulation of the trpEDCBA operon and the gene pheA is also controlled by a system called attenuation. In the case of the trp operon this was first discovered when it was observed that mutants with internal deletions, but that still retained the trp operator region intact, since they were still subject to repression by tryptophan, had enhanced levels of expression of the trpC, -B, and -A genes (170). Subsequent detailed investigations revealed this to be a consequence of the negation of a second transcriptional control, which, in the wild-type was causing premature transcriptional termination of the majority of the transcripts before any of the pathway genes had been transcribed. This process was termed attenuation and, in the case of the trp operon, it senses the availability of charged tryptophanyl-t-RNA. By expressing the lacZ gene separately from the trp promoter/operator in a construct lacking the attenuator, Yanofsky was able to measure the effects of TrpR-mediated repression in the absence of attenuation and compare that with the combined effects of repression and attenuation. By using selected bradytrophs to starve cells for tryptophan, it was possible to show that attenuation was not relaxed until after the point where tryptophan starvation was sufficient to relieve repression (374).
The process of attenuation has been the subject of numerous very detailed reviews (196, 198, 367). In brief, in the case of tryptophan, the transcript of the tryptophan operon contains a leader sequence that has the capacity to assume two alternate secondary structures and that also encodes a leader peptide. One of these RNA structures causes termination of transcription before RNA polymerase reaches the trpE-coding sequence and the other does not. The leader peptide contains two adjacent tryptophan codons so positioned that if a ribosome traveling behind the RNA polymerase stalls at this position because of an extreme scarcity of charged tRNATrp, it prevents the base pairing necessary for the formation of the transcription termination structure and transcription continues into the various genes of the operon. When there is ample charged tRNATrp, the ribosomes do not stall and the termination structure of the RNA is able to form. The termination and antitermination structures of the leader sequence are shown in Fig. 10.
To understand why E. coli has evolved such complex systems for regulating the synthesis and activity of the tryptophan pathway enzymes, Yanofsky and Horn (372) have carried out a series of experiments involving various regulatory mutants and shifts between growth media with high and low levels of tryptophan. From these studies they conclude that the sum of all the regulatory circuits are required to ensure that growth can continue at an unreduced rate when the organism is shifted from a tryptophan-rich environment to one where maximal endogenous synthesis is required. Computer simulations have also been carried out to evaluate the role of these different systems and the feedback sensitivity of anthranilate synthase in maintaining adequate levels of synthesis under different conditions (36, 285).
The expression of the pheA gene is controlled by a system of transcriptional attenuation that senses the availability of charged phenylalanyl-t-RNAPhe (384). Early reports that appeared to identify operator mutations in pheA and unlinked mutations in a putative regulator pheR have now been explained as mutations in the leader region of the pheA gene or as mutations affecting the availability of charged phenylalanyl-t-RNAPhe (114, 115, 116, 129, 134, 135, 265). The role of attenuation in controlling expression of the pheA gene is also extensively discussed in various general reviews (196).
Work carried out in the authors’ laboratory was supported by the Australian Research Committee.
References
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Module3.6.1.8
