Indole-3-glycerol Phosphate Synthase Research Articles

The hyperthermostable indoleglycerol phosphate synthase from Thermotoga maritima is destabilized by mutational disruption of two solvent-exposed salt bridges

The recombinantly expressed protein indoleglycerol phosphate synthase from the hyperthermophilic bacterium Thermotoga maritima (tIGPS) was purified and characterized with respect to oligomerization state, catalytic properties and thermostability. This enzyme from the biosynthetic pathway of tryptophan is a monomer in solution. In contrast to IGPS from the hyperthermophilic archaeon Sulfolobus solfataricus, tIGPS shows high catalytic activity at room temperature and only weak product inhibition. In order to test the hypothesis that salt bridges in a critical context contribute to the high thermostability of tIGPS, two solvent-exposed salt bridges were selected, based on its three-dimensional structure, for individual disruption by site-directed mutagenesis. The first salt bridge fixes the N terminus to the core of the protein, and the second serves as a clamp between helices α1 and α8, which are widely separated in sequence but adjacent in the (βα)8-barrel. Kinetics of irreversible heat inactivation reveal that the salt bridge crosslinking helices α1 and α8 stabilizes tIGPS more strongly than that tethering the N terminus.

Involvement of Phenazines and Anthranilate in the Antagonism of Pseudomonas aeruginosa PNA1 and Tn5 Derivatives Toward Fusarium spp. and Pythium spp.

Pseudomonas aeruginosa PNA1, isolated from the rhizosphere of chickpea in India, suppressed Fusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris, and Pythium damping-off of bean, caused by Pythium splendens. When grown in culture, PNA1 produced the phenazine antibiotics phenazine-1-carboxylic acid and oxychloraphine, and inhibited mycelial growth of F. oxysporum f. sp. ciceris, P. splendens, and certain other phytopathogenic fungi. Two mutants (FM29 and FM13) deficient in phenazine production were obtained following transposon mutagenesis of PNA1. The transposon in the genome of FM29 was localized to phnA, which is thought to encode a subunit of anthranilate synthase II involved in the phenazine biosynthesis. The FM13 mutation was complemented by trpC, which encodes indole glycerol phosphate synthase in the tryptophan biosynthesis pathway; consequently, FM13 could not grow on a minimal medium in the absence of tryptophan. Neither FM29 nor FM13 suppressed Fusarium wilt of chickpea to the level achieved by the wild-type strain, indicating that phenazine production contributed to the biocontrol of this disease by P. aeruginosa PNA1. FM29 was also less effective than the phenazine-producing parental strain in biological control of Pythium damping-off of bean, but FM13 was as effective as the parental strain in suppressing this disease. Anthranilate, an intermediate in the tryptophan biosynthesis pathway, suppressed mycelial growth of Pythium spp. in culture and Pythium damping-off of bean and lettuce. Anthranilate, excreted by FM13 as a consequence of the trpC mutation, may have contributed to the suppression of Pythium damping-off by the mutant.

Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli.

Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5'-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184.

Nonsequential unfolding of the alpha/beta barrel protein indole-3-glycerol-phosphate synthase.

The folding of the enzyme indole-3-glycerol-phosphate synthase (IGPS), a member of the (alpha/beta)8 fold family, has been studied. At least two folding intermediates have been detected using spectroscopic and activity measurements in combination with gel filtration chromatography. These two intermediates are produced by parallel pathways of a nonsequential unfolding mechanism rather than being consecutive steps in a sequential process. One intermediate can be detected in unfolding experiments because it is kinetically trapped in that conformation, but it is not observed in refolding experiments. It has spectroscopic and hydrodynamic properties very similar to those of the native protein, but it is inactive. The other intermediate could not be characterized because it either aggregates or unfolds under our experimental conditions and could not be isolated chromatographically.

Deletion mutagenesis as a test of evolutionary relatedness of indoleglycerol phosphate synthase with other TIM barrel enzymes

The role of the extra helix α 0 in the N-terminal extension of the eight-fold βα barrel of indoleglycerol phosphate synthase was probed by point mutation and truncation. Replacing invariant leucine 5 by valine of the enzyme from Escherichia coli affected neither k cat nor K M, but deletion of 8 N-terminal residues decreased solubility strongly. The similarly truncated variant from the hyperthermophile Sulfolobus solfataricus was soluble, and had the same k cat value as the wild-type protein but a 220-fold greater K M value. These results suggest that the N-terminal portion of helix α 0 provides for strong binding of the substrate, but is not essential for stabilizing the bound transition state. Thus, three enzymes of tryptophan biosynthesis operate essentially as canonical eight-fold βα barrels, as required for their divergent evolution. © 1997 Federation of European Biochemical Societies.

Assessment of a protein fold recognition method that takes into account four physicochemical properties: side-chain packing, solvation, hydrogen-bonding, and local conformation.

A protein fold recognition method was tested by the blind prediction of the structures of a set of proteins. The method evaluates the compatibility of an amino acid sequence with a three-dimensional structure using the four evaluation functions: side-chain packing, solvation, hydrogen-bonding, and local conformation functions. The structures of 14 proteins containing 19 sequences were predicted. The predictions were compared with the experimental structures. The experimental results showed that 9 of the 19 target sequences have known folds or portions of known folds. Among them, the folds of Klebsiella aerogenes urease beta subunit (KAUB) and pyruvate phosphate dikinase domain 4 (PPDK4) were successfully recognized; our method predicted that KAUB and PPDK4 would adopt the folds of macromomycin (Ig-fold) and phosphoribosylanthranilate isomerase:indoleglycerol-phosphate synthase (TIM barrel), respectively, and the experimental structure revealed that they actually adopt the predicted folds. The predictions for the other targets were not successful, but they often gave secondary structural patterns similar to those of the experimental structures.

(Beta alpha)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima.

To better understand the evolution of a key metabolic pathway, we have sequenced the trpCFBA gene cluster of the hyperthermophilic bacterium Thermotoga maritima. The genes were cloned by complementation in vivo of trp deletion strains of Escherichia coli. The new sequences, together with earlier findings, establish that the trp operon of T.maritima has the order trpE(G.D)CFBA, which might represent the ancestral organization of the tryptophan operon. Heterologous expression of the trp(G.D) and trpC genes in E.coli and N-terminal sequencing of their polypeptide products showed that their translation is initiated at the rate start codons TTG and ATC, respectively. Consequently, the N-terminus of the trp(G.D) fusion protein is 43 residues shorter than previously postulated. Amino acid composition and sequence analyses of the protein products of T.maritima trpC (indoleglycerol phosphate synthase), trpF (phosphoribosyl anthranilate isomerase) and trpA (alpha-subunit of tryptophan synthase) suggest that these thermostable (beta alpha)8-barrel proteins may be stabilized by additional salt bridges, compared with the mesostable forms. Another notable feature is the predicted lack of the N-terminal helix alpha 0 in the alpha-subunit of tryptophan synthase.

Symbiotic ineffectiveness of trpCD deletion mutants of Bradyrhizobium japonicum

Genes responsible for tryptophan biosynthesis are thought to be essential for symbiotic competence of Bradyrhizobium japonicum. The B. japonicum trpCD region, cloned as a 2.8-kb HindIII- BamHI fragment, had an internal 1.2-kb SmaI fragment that was deleted and replaced in vitro with interposon omega carrying a gene specifying streptomycin and spectinomycin resistance. Site-directed marker exchange mutagenesis of the B. japonicum genome was performed in vivo and the construction of deletion mutants was confirmed by Southern blot analysis of the DNA of the exconjugants. Since the trpCD deletion mutants grew on indole they evidently were unaffected in expression of trpAB and we interpret these data as indicating that the organization of the genes for tryptophan biosynthesis in B. japonicum differs from that of enterics. The new Trp − deletion mutants lacked activities for the trpC- and trpD-encoded enzymes, indole glycerol phosphate synthase (EC 4.1.1.48) and phosphoribosyl anthranilate transferase (EC 2.4.2.18), respectively, but had a higher specific activity for the trpEG-encoded enzyme, anthranilate synthase (EC 4.1.3.27). The new trpCD deletion mutants failed to effectively nodulate soybean. Thus, we conclusively demonstrate the essential role of trpCD in symbiosis.

Divergent evolution of a beta/alpha-barrel subclass: detection of numerous phosphate-binding sites by motif search.

Study of the most conserved region in many beta/alpha-barrels, the phosphate-binding site, revealed a sequence motif in a few beta/alpha-barrels with known tertiary structure, namely glycolate oxidase (GOX), cytochrome b2 (Cyb2), tryptophan synthase alpha subunit (TrpA), and the indoleglycerolphosphate synthase (TrpC). Database searches identified this motif in numerous other enzyme families: (1) IMP dehydrogenase (IMPDH) and GMP reductase (GuaC); (2) phosphoribosylformimino-5-aminoimidazol carboxamide ribotide isomerase (HisA) and the cyclase-producing D-erythro-imidazole-glycerolphosphate (HisF) of the histidine biosynthetic pathway; (3) dihydroorotate dehydrogenase (PyrD); (4) glutamate synthase (GltB); (5) ThiE and ThiG involved in the biosynthesis of thiamine as well as related proteins; (6) an uncharacterized open reading frame from Erwinia herbicola; and (7) a glycerol uptake operon antiterminator regulatory protein (GlpP). Secondary structure predictions of the different families mentioned above revealed an alternating order of beta-strands and alpha-helices in agreement with a beta/alpha-barrel-like topology. The putative phosphate-binding site is always found near the C-terminus of the enzymes, which are all at least about 200 amino acids long. This is compatible with its assumed location between strand 7 and helix 8. The identification of a significant motif in functionally diverse enzymes suggests a divergent evolution of at least a considerable fraction of beta/alpha-barrels. In addition to the known accumulation of beta/alpha-barrels in the tryptophan biosynthetic pathway, we observe clusters of these enzymes in histidine biosynthesis, purine metabolism, and apparently also in thiamine biosynthesis. The substrates are mostly heterocyclic compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein folding within the cell is influenced by controlled rates of polypeptide elongation

Previous studies have proposed that specific translational pauses have evolved to promote protein folding inside the cell by temporally separating the folding of specific regions of some polypeptide chains during their synthesis. Here we show that this is the case for a bifunctional protein in Saccharomyces cerevisiae. The yeast TRP3 gene contains a translational pause comprising ten contiguous non-preferred codons within its second functional domain (indoleglycerol phosphate synthase). Site-directed mutagenesis was used to remove this translational pause by increasing the codon bias of the region without changing the amino acid sequence of the protein (to create the gene TRP3pr: pause replaced). The TRP3pr gene was able to complement a trp3 :: URA3 null mutation in yeast. No significant differences in the doubling times of TRP3 or TRP3 pr yeast transformants were observed during growth at 25 °C, 30 °C or 37 °C, or in the presence of sublethal concentrations of the analogue, 5-methyltryptophan. However, further analysis of TRP3 and TRP3pr transformants revealed that the removal of the translational pause causes a 1.5-fold decrease in indoleglycerol phosphate synthase activity per TRP3 mRNA. This observation which is statistically significant ( P < 0.05) and reproducible, suggests that translational pausing promotes the correct intracellular folding of the TRP3 protein.

Sequence of the indoleglycerol phosphate synthase (trpC) gene from Rhodobacter capsulatus.

We have isolated, cloned, and sequenced the indoleglycerol phosphate synthase gene (trpC) from Rhodobacter capsulatus. Normalized alignment scores comparing the trpC gene of R. capsulatus with the trpC genes of other bacterial species are reported. An unexpected degree of similarity to the trpC gene of Bacillus subtilis was found.

Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: Indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 Å 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 Å 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 Å resolution. The root-mean-square deviations from ideal bond lengths and bond angles are 0·010 Å and 3·2 °, respectively. The structure of N-(5′-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E. coli comprises two β α -barrel domains that superimpose with a root-mean-square deviation of 2·03 Å for 138 C α-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 β-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 β α -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 β α -barrel ancestor.

Structural conservation in parallel .beta./.alpha.-barrel enzymes that catalyze three sequential reactions in the pathway of tryptophan biosynthesis

Three successive steps in tryptophan biosynthesis are catalyzed by single-domain proteins, each folded as a parallel beta/alpha-barrel, as observed in the crystal structures of the bienzyme (phosphoribosyl)-anthranilate isomerase:indoleglycerolphosphate synthase from Escherichia coli [Priestle, J.P., Grütter, M. G., White, J. L., Vincent, M. G., Kania, M., Wilson, E., Jardetzky, T. S., Kirschner, K., & Jansonius, J. N. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5690-5694] and the alpha-subunit of the tetrameric bienzyme tryptophan synthase from Salmonella typhimurium [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Recent refinement of the crystal structures of these enzymes at atomic resolution revealed that they contain a common phosphate group binding site in the beta/alpha-barrel, created by residues of the loop between beta-strand 7 and alpha-helix 7 and the N-terminus of an additional helix 8'. The close similarities of their beta/alpha-barrel structures permitted the alignment of 50-75% of their respective amino acid sequences. Considerable sequence similarity was detected in the regions spanning the phosphate binding sites, whereas the percentage of identical residues was barely significant for the remaining parts of the enzymes. These observations suggest divergent evolution of these three beta/alpha-barrel enzymes involved in tryptophan biosynthesis. The same phosphate binding site was also observed in six other beta/alpha-barrel enzymes that are functionally unrelated to those involved in tryptophan biosynthesis: triosephosphate isomerase, ribulose-1,5-bisphosphate carboxylase/oxygenase, glycolate oxidase, flavocytochrome b2, trimethylamine dehydrogenase, and tentatively also fructosebisphosphate aldolase.(ABSTRACT TRUNCATED AT 250 WORDS)

Deletion analysis of domain independence in the TRP1 gene product of Neurospora crassa

The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5'-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.

Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus.

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.

Modification of a catalytically important residue of indoleglycerol-phosphate synthase from Escherichia coli

The active-site residues of indoleglycerol-phosphate synthase from Escherichia coli were tentatively localized by comparing crystallographic data with the amino acid identities among the known indoleglycerol-phosphate synthase sequences. To test the validity of the resulting model of catalysis one of the residues in the presumptive active site, Lys 55, was changed to serine using oligonucleotide-directed mutagenesis. The specificity constant k cat/ K m of the mutant is 3 × 10 4-times lower than that of the wild-type enzyme, due to a 60-fold decrease in k cat and a 450-fold increase in K m. This finding shows that Lys 55 is important for both catalysis and substrate binding.

The role of the TRP1 gene in yeast tryptophan biosynthesis.

Transcription of the gene for phosphoribosyl-anthranilate isomerase (TRP1) from the TRP1 promoter is initiated only approximately half as frequently as, for example, from the TRP3 promoter, but TRP1 mRNA is approximately twice as stable as TRP3 mRNA. Therefore, the steady state amount of TRP1 mRNA in yeast cells, grown without amino acid limitation, is similar to the steady-state amount of TRP3 mRNA. The protein concentration of both enzymes in yeast cells is about the same, but the basal specific enzyme activity in permeabilized cells of the TRP1 gene product N-(5'-phosphoribosyl-1)-anthranilate isomerase is about 2-3 times higher than that of any of the other TRP enzymes. According to the kinetic parameters of the purified isomerase protein, the enzyme is more active than, for example, the purified TRP3 enzyme indoleglycerol-phosphate synthase. It is suggested that the TRP1 gene of Saccharomyces cerevisiae might be the result of a rearrangement event, separating the N-(5'-phosphoribosyl-1)-anthranilate isomerase domain from the indoleglycerol-phosphate synthase domain and putting the catalytically more active isomerase domain behind a weak and nonregulated constitutive promoter.

Isolation of auxotrophs and analysis of regulation of tryptophan biosynthesis in Zymomonas mobilis

Tryptophan auxotrophs were isolated and used to analyze the regulation of tryptophan biosynthesis in Zymomonas mobilis. Twelve tryptophan auxotrophs were cassified as trp E, B or A based on accumulation of, or growth on, indole and anthranilic acid. Trp B mutants were found to accumulate indole when grown on limiting, but not on excess tryptophan, suggesting that tryptophan plays a role in regulating its biosynthesis. Tryptophan synthase and indoleglycerol phosphate synthase specific activities were measured in the wild-type strain and two trp mutants grown in limiting or excess tryptophan. Neither activity was repressed by exogenous tryptophan.

48] Phosphoribosylanthranilate isomerase—indoleglycerol-phosphate synthase from Escherichia coli

48] Phosphoribosylanthranilate isomerase—indoleglycerol-phosphate synthase from Escherichia coli

A cloned tryptophan-synthesis gene from the Ascomycete Cochliobolus heterostrophus functions in Escherichia coli, yeast and Aspergillus nidulans

A gene (TRP1) in the tryptophan biosynthetic pathway of the fungal plant pathogen Cochliobolus heterostrophus was isolated by complementation of an Escherichia coli trpF mutant which lacked phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene also complemented an E. coli trpC mutant lacking indoleglycerolphosphate synthase (IGPS) activity, a yeast trp1 mutant missing PRAI activity and an Aspergillus nidulans trpC mutant. It functioned in E. coli and A. nidulans without apparent rearrangement but in yeast only after the 5' end of the gene was deleted. The gene was subcloned on a 4.65-kb DNA fragment and the PRAI domain was localized to a 2.9-kb region. It showed homology to the A. nidulans trpC and Neurospora crassa trp-1 genes. There was one predominant transcript of C. heterostrophus TRP1, the same size (2.6-kb) as one of the two functional transcripts produced by A. nidulans trpC. The constitutive activity of the C. heterostrophus TRP1 gene was high whereas that of the A. nidulans trpC gene was low.