Mutations in the trpD gene of Corynebacterium glutamicum confer 5-methyltryptophan resistance by encoding a feedback-resistant anthranilate phosphoribosyltransferase.

The trpD gene from tryptophan-hyperproducing Corynebacterium glutamicum ATCC 21850 was isolated on the basis of its ability to confer resistance to 5-methyltryptophan on wild-type C. glutamicum AS019. Comparative sequence analysis of the genes from the wild-type AS019 and ATCC 21850 trpD genes revealed two amino acid substitutions at the protein level. Further analysis demonstrated that the trpD gene product from ATCC 21850, anthranilate phosphoribosyltransferase, was more resistant to feedback inhibition by either tryptophan or 5-methyltryptophan than its wild-type counterpart. It is proposed that phosphoribosyltransferase insensitivity to tryptophan in ATCC 21850 contributes to an elevated level of tryptophan biosynthesis.     

The structure of MbtI from Mycobacterium tuberculosis, the first enzyme in the biosynthesis of the siderophore mycobactin, reveals it to be a salicylate synthase

The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. Mycobacterium tuberculosis acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for Pseudomonas aeruginosa, or a single-step conversion catalyzed by a salicylate synthase, as with Yersinia enterocolitica. Here we present the structure of the enzyme MbtI (Rv2386c) from M. tuberculosis, solved by multiwavelength anomalous diffraction at a resolution of 1.8 A, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg2+ for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from Y. enterocolitica and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in M. tuberculosis.     

Tryptophan of facultative methylotrophic bacteria Pseudomonas sp. M. II. Regulation of tryptophan biosynthesis

Regulation of tryptophan biosynthesis of facultative methylotrophic Pseudomonas sp. M was studied. Repression of the trpE, trpD and trpC genes by tryptophan was demonstrated. It was also shown that the trpE and trpDC genes are derepressed noncoordinately. No regulation of the trpF gene product could be demonstrated, indicating that its synthesis is constitutive. The trpA and trpB genes are inducible by indol-3-glycerophosphate. Anthranilate synthase and tryptophan synthase were sensitive to the feedback inhibition. The tryptophan concentrations giving 50% inhibition were estimated to be 9 microM and 1 microM, respectively. Experimental evidence for activation of the N-5-phosphoribosyl anthranilate isomerase and for inhibition of the indol-3-glycerophosphate synthase by some tryptophan intermediates was obtained.     

Regulation of tryptophan genes in Rhizobium leguminosarum.

Twelve tryptophan auxotrophs of Rhizobium leguminosarum were characterized biochemically. They were grown in complex and minimal media with several carbon sources, in both limiting and excess tryptophan. Missing enzyme activities allowed assignment of all mutant to the trpE, trpD, trpB, or trpA gene, confirming earlier results with the same mutants (Johnston et al., Mol. Gen. Genet. 165:323-330, 1978). In regulatory experiments, only the first enzyme of the pathway, anthranilate synthase, responded (about 15-fold) to tryptophan excess or limitation.     

Crystal structure of thiamindiphosphate-dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid.

The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 A resolution and refined to a crystallographic R-factor of 20.5% (Rfree 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open alpha/beta topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis, but with a relative difference of 20 degrees in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan.     

Structural analysis of two enzymes catalysing reverse metabolic reactions implies common ancestry

The crystal structure of the dimeric anthranilate phosphoribosyltransferase (AnPRT) reveals a new category of phosphoribosyltransferases, designated as class III. The active site of this enzyme is located within the flexible hinge region of its two-domain structure. The pyrophosphate moiety of phosphoribosylpyrophosphate is co-ordinated by a metal ion and is bound by two conserved loop regions within this hinge region. With the structure of AnPRT available, structural analysis of all enzymatic activities of the tryptophan biosynthesis pathway is complete, thereby connecting the evolution of its enzyme members to the general development of metabolic processes. Its structure reveals it to have the same fold, topology, active site location and type of association as class II nucleoside phosphorylases. At the level of sequences, this relationship is mirrored by 13 structurally invariant residues common to both enzyme families. Taken together, these data imply common ancestry of enzymes catalysing reverse biological processes--the ribosylation and deribosylation of metabolic pathway intermediates. These relationships establish new links for enzymes involved in nucleotide and amino acid metabolism.     

Crystallization and structure solution at 4 A resolution of the recombinant synthase domain of N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli complexed to a substrate analogue

The recombinant synthase domain of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been crystallized, and the structure has been solved at 4 A resolution. Two closely related crystal forms grown from ammonium sulphate diffract to 2 A resolution. One form (space group R32, a = 163 A, alpha = 29.5 degrees) contains the unliganded synthase domain; the second crystal form (space group P6(3)22, a = 144 A, c = 158 A) is co-crystallized with the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate. The structure of the synthase-inhibitor complex has been solved by the molecular replacement method. This achievement represents the first successful use of a (beta alpha)8-barrel monomer as a trial model. The recombinant synthase domain associates as a trimer in the crystal, the molecules being related by a pseudo-crystallographic triad. The interface contacts between the three domains are mediated by those residues that are also involved in the domain interface of the bifunctional enzyme. This system provides a model for an interface which is used in both intermolecular and intramolecular domain contacts.     

Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution.

The three-dimensional structure of the monomeric bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been refined at 2.0 A resolution, using oscillation film data obtained from synchrotron radiation. The model includes the complete protein (452 residues), two phosphate ions and 628 water molecules. The final R-factor is 17.3% for all observed data between 15 and 2 A resolution. The root-mean-square deviations from ideal bond lengths and bond angles are 0.010 A and 3.2 degrees, respectively. The structure of N-(5'-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E. coli comprises two beta/alpha-barrel domains that superimpose with a root-mean-square deviation of 2.03 A for 138 C alpha-pairs. The C-terminal domain (residues 256 to 452) catalyses the PRAI reaction and the N-terminal domain (residues 1 to 255) catalyses the IGPS reaction, two sequential steps in tryptophan biosynthesis. The enzyme has the overall shape of a dumb-bell, resulting in a surface area that is considerably larger than normally observed for monomeric proteins of this size. The active sites of the PRAI and the IGPS domains, both located at the C-terminal side of the central beta-barrel, contain equivalent binding sites for the phosphate moieties of the substrates N-(5'-phosphoribosyl) anthranilate and 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate. These two phosphate binding sites are identical with respect to their positions within the tertiary structure of the beta/alpha-barrel, the conformation of the residues involved in phosphate binding and the hydrogen-bonding network between the phosphate ions and the protein. The active site cavities of both domains contain similar hydrophobic pockets that presumably bind the anthranilic acid moieties of the substrates. These similarities of the tertiary structures and the active sites of the two domains provide evidence that N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from E. coli results from a gene duplication event of a monomeric beta/alpha-barrel ancestor.     

Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway.

Arabidopsis thaliana has two genes, ASA1 and ASA2, encoding the alpha subunit of anthranilate synthase, the enzyme catalyzing the first reaction in the tryptophan biosynthetic pathway. As a branchpoint enzyme in aromatic amino acid biosynthesis, anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved amino acids involved in feedback inhibition by tryptophan. ASA1 and ASA2 cDNAs complement anthranilate synthase alpha subunit mutations in the yeast Saccharomyces cerevisiae and in Escherichia coli, confirming that both genes encode functional anthranilate synthase proteins. The distributions of ASA1 and ASA2 mRNAs in various parts of Arabidopsis plants are overlapping but nonidentical, and ASA1 mRNA is approximately 10 times more abundant in whole plants. Whereas ASA2 is expressed at a constitutive basal level, ASA1 is induced by wounding and bacterial pathogen infiltration, suggesting a novel role for ASA1 in the production of tryptophan pathway metabolites as part of an Arabidopsis defense response. Regulation of key steps in aromatic amino acid biosynthesis in Arabidopsis appears to involve differential expression of duplicated genes.     

DNA sequences and characterization of four early genes of the tryptophan pathway in Pseudomonas aeruginosa.

Two pairs of related but easily distinguishable genes for the two subunits of anthranilate synthase have been identified in Pseudomonas aeruginosa. These were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance cassette, and returned to the P. aeruginosa chromosome, replacing the wild-type gene. Gene replacement implicated only one of the pairs in tryptophan biosynthesis. This report describes the cloning and sequencing of the tryptophan-related gene pair, designated trpE and trpG, and presents experiments implicating their gene products in tryptophan production. DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also includes trpD and trpC. Complementation of Trp auxotrophs by R-prime plasmids (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983) has shown that a large cluster of pyocin R2 genes is flanked at one end by trpE and the other end by trpDC; the physical map that was obtained shows the distance between trpE and trpDC to be about 25 kilobases. Our restriction map of the trpE and trpGDC regions agrees with data presented by Shinomiya et al.     

Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC.

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.     

Structure and mechanism of MbtI, the salicylate synthase from Mycobacterium tuberculosis

MbtI (rv2386c) from Mycobacterium tuberculosis catalyzes the initial transformation in mycobactin biosynthesis by converting chorismate to salicylate. We report here the structure of MbtI at 2.5 A resolution and demonstrate that isochorismate is a kinetically competent intermediate in the synthesis of salicylate from chorismate. At pH values below 7.5 isochorismate is the dominant product while above this pH value the enzyme converts chorismate to salicylate without the accumulation of isochorismate in solution. The salicylate and isochorismate synthase activities of MbtI are Mg2+-dependent, and in the absence of Mg2+ MbtI has a promiscuous chorismate mutase activity similar to that of the isochorismate pyruvate lyase, PchB, from Pseudomonas aeruginosa. MbtI is part of a larger family of chorismate-binding enzymes descended from a common ancestor (the MST family), that includes the isochorismate synthases and anthranilate synthases. The lack of active site residues unique to pyruvate eliminating members of this family, combined with the observed chorismate mutase activity, suggests that MbtI may exploit a sigmatropic pyruvate elimination mechanism similar to that proposed for PchB. Using a combination of structural, kinetic, and sequence based studies we propose a mechanism for MbtI applicable to all members of the MST enzyme family.     

Tryptophan synthetase in Euglena gracilis strain G

The five enzyme activities in the synthesis of l-tryptophan have been obtained in extracts of Euglena gracilis. One of these, tryptophan synthetase, has been studied in detail. The general catalytic properties of tryptophan synthetase, including the range of reactions catalyzed and its substrate and cofactor affinities, are similar to those reported for other organisms. The Euglena enzyme has two properties never previously observed for tryptophan synthetase. First, the rate of catalysis of the conversion of indole-glycerol phosphate to l-tryptophan remained at its maximal value and was unaffected by the ionic environment up to 0.3 m KCl. In contrast, the conversion of indole to tryptophan showed a sharp maximum at 0.08 m KCl. Second, the enzyme is a component of a complex that includes every enzyme in the pathway committed to tryptophan biosynthesis with the exception of anthranilate synthetase, the regulatory enzyme.     

Wild-type phosphoribosylpyrophosphate synthase (PRS) from Mycobacterium tuberculosis: a bacterial class II PRS?

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme.     

Enzymatic Basis for Overproduction of Tryptophan and Its Metabolites in Hansenula polymorpha Mutants

3-Deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthetase and anthranilate synthetase are key regulatory enzymes in the aromatic amino acid biosynthetic pathway. The DAHP synthetase activity of Hansenula polymorpha was subject to additive feedback inhibition by phenylalanine and tyrosine but not by tryptophan. The synthesis of DAHP synthetase in this yeast was not repressed by exogenous aromatic amino acids, singly or in combinations. The activity of anthranilate synthetase was sensitive to feedback inhibition by tryptophan, but exogenous tryptophan did not repress the synthesis of this enzyme. Nevertheless, internal repression of anthranilate synthetase probably exists, since the content of this enzyme in H. polymorpha strain 3-136 was double that in the wild-type and less sensitive 5-fluorotryptophan-resistant strains. The biochemical mechanism for the overproduction of indoles by the 5-fluorotryptophan-resistant mutants was due primarily to a partial desensitization of the anthranilate synthetase of these strains to feedback inhibition by tryptophan. These results support the concept that inhibition of enzyme activities rather than enzyme repression is more important in the regulation of aromatic amino acid biosynthesis in H. polymorpha.     

Mapping of the tryptophan genes of Acinetobacter calcoaceticus by transformation

Auxotrophs of Acinetobacter calcoaceticus blocked in each reaction of the synthetic pathway from chorismic acid to tryptophan were obtained after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. One novel class was found to be blocked in both anthranilate and p-aminobenzoate synthesis; these mutants (trpG) require p-aminobenzoate or folate as well as tryptophan (or anthranilate) for growth. The loci of six other auxotrophic classes requiring only tryptophan were defined by growth, accumulation, and enzymatic analysis where appropriate. The trp mutations map in three chromosomal locations. One group contains trpC and trpD (indoleglycerol phosphate synthetase and phosphoribosyl transferase) in addition to trpG mutations; this group is closely linked to a locus conferring a glutamate requirement. Another cluster contains trpA and trpB, coding for the two tryptophan synthetase (EC 4.2.1.20) subunits, along with trpF (phosphoribosylanthranilate isomerase); this group is weakly linked to a his marker. The trpE gene, coding for the large subunit of anthranilate synthetase, is unlinked to any of the above. This chromosomal distribution of the trp genes has not been observed in other organisms.     

Localization of Two Functions of the Phosphoribosyl Anthranilate Transferase of Escherichia coli to Distinct Regions of the Polypeptide Chain

The trpD gene specifies a polypeptide which has both glutamine amidotransferase and phosphoribosyl anthranilate (PRA) transferase activities. Deletions fusing segments of trpD to the gene preceding it in the operon, trpE, were selected in strains carrying various trpD point mutations. The selection procedure required both that a deletion enter trpE and that it restore the PRA transferase activity which the parent trpD point mutant lacked. Deletion mutants were found which had PRA transferase activity although the first third of trpD was deleted. The existence of the mutants proves that a terminal segment of trpD is sufficient to specify a polypeptide having PRA transferase activity. The location of the deletion end points on the genetic map of trpD defines the extent of the trpD segment required for PRA transferase activity. This segment did not overlap the initial region of trpD required to specify the glutamine amidotransferase function of the trpD polypeptide. These results support the hypothesis (M. Grieshaber and R. Bauerle, 1972; H. Zalkin and L. H. Hwang, 1971) that the bifunctional trpD polypeptide might have evolved by fusion of a gene specifying a glutamine amidotransferase with a gene directing PRA transferase synthesis.