Structure of Escherichia coli aminodeoxychorismate synthase: architectural conservation and diversity in chorismate-utilizing enzymes

Aminodeoxychorismate synthase is part of a heterodimeric complex that catalyzes the two-step biosynthesis of 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folate in microorganisms. In the first step, a glutamine amidotransferase encoded by the pabA gene generates ammonia as a substrate that, along with chorismate, is used in the second step, catalyzed by aminodeoxychorismate synthase, the product of the pabB gene. Here we report the X-ray crystal structure of Escherichia coli PabB determined in two different crystal forms, each at 2.0 A resolution. The 453-residue monomeric PabB has a complex alpha/beta fold which is similar to that seen in the structures of homologous, oligomeric TrpE subunits of several anthranilate synthases of microbial origin. A comparison of the structures of these two classes of chorismate-utilizing enzymes provides a rationale for the differences in quaternary structures seen for these enzymes, and indicates that the weak or transient association of PabB with PabA during catalysis stems at least partly from a limited interface for protein interactions. Additional analyses of the structures enabled the tentative identification of the active site of PabB, which contains a number of residues implicated from previous biochemical and genetic studies to be essential for activity. Differences in the structures determined from phosphate- and formate-grown crystals, and the location of an adventitious formate ion, suggest that conformational changes in loop regions adjacent to the active site may be needed for catalysis. A surprising finding in the structure of PabB was the presence of a tryptophan molecule deeply embedded in a binding pocket that is analogous to the regulatory site in the TrpE subunits of the anthranilate synthases. The strongly bound ligand, which cannot be dissociated without denaturation of PabB, may play a structural role in the enzyme since there is no effect of tryptophan on the enzymic synthesis of aminodeoxychorismate. Extensive sequence similarity in the tryptophan-binding pocket among several other chorismate-utilizing enzymes, including isochorismate synthase, suggests that they too may bind tryptophan for structural integrity, and corroborates early ideas on the evolution of this interesting enzyme family.     

Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications.

Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.     

Regulation of a Ligand-Mediated Association-Dissociation System of Anthranilate Synthesis in Clostridium butyricum

The anthranilate synthetase of Clostridium butyricum is composed of two nonidentical subunits of unequal size. An enzyme complex consisting of both subunits is required for glutamine utilization in the formation of anthranilic acid. Formation of anthranilate will proceed in the presence of partially pure subunit I provided ammonia is available in place of glutamine. Partially pure subunit II neither catalyzes the formation of anthranilate nor possesses anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity. The enzyme complex is stabilized by high subunit concentrations and by the presence of glutamine. High KCl concentrations promote dissociation of the enzyme into its component subunits. The synthesis of subunits I and II is coordinately controlled with the synthesis of the enzymes mediating reactions 4 and 5 of the tryptophan pathway. When using gel filtration procedures, the molecular weights of the large (I) and small (II) subunits were estimated to be 127,000 and 15,000, respectively. Partially pure anthranilate synthetase subunits were obtained from two spontaneous mutants resistant to growth inhibition by 5-methyltryptophan. One mutant, strain mtr-8, possessed an anthranilate synthetase that was resistant to feedback inhibition by tryptophan and by three tryptophan analogues: 5-methyl-tryptophan, 4- and 5-fluorotryptophan. Reconstruction experiments carried out by using partially purified enzyme subunits obtained from wild-type, mutant mtr-8 and mutant mtr-4 cells indicate that resistance of the enzyme from mutant mtr-8 to feedback inhibition by tryptophan or its analogues was the result of an alteration in the large (I) subunit. Mutant mtr-8 incorporates [(14)C]tryptophan into cell protein at a rate comparable with wild-type cells. Mutant mtr-4 failed to incorporate significant amounts of [(14)C]tryptophan into cell protein. We conclude that strain mtr-4 is resistant to growth inhibition by 5-methyltryptophan because it fails to transport the analogue into the cell. Although mutant mtr-8 was isolated as a spontaneous mutant having two different properties (altered regulatory properties and an anthranilate synthetase with altered sensitivity to feedback inhibition), we have no direct evidence that this was the result of a single mutational event.     

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the alpha subunits OASA1 and OASA2

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, alpha and beta, and converts chorismate to anthranilate. Two or more AS alpha-subunit genes have been identified and characterized in several land plants. Although alpha subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS alpha-subunit genes (OASA1 and OASA2) in rice (Oryza sativa ). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS beta subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two beta subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a beta subunit to achieve maximal enzymatic activity even with NH(4)(+) as the amino donor. The V (max) and K (i) for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.     

Anthranilate synthase from Ruta graveolens. Duplicated AS alpha genes encode tryptophan-sensitive and tryptophan-insensitive isoenzymes specific to amino acid and alkaloid biosynthesis.

Anthranilate synthase (AS, EC 4.1.3.27) catalyzes the conversion of chorismate into anthranilate, the biosynthetic precursor of both tryptophan and numerous secondary metabolites, including inducible plant defense compounds. The higher plant Ruta graveolens produces tryptophan and elicitor-inducible, anthranilate-derived alkaloids by means of two differentially expressed nuclear genes for chloroplast-localized AS alpha subunits, AS alpha 1 and AS alpha 2. Mechanisms that partition chorismate between tryptophan and inducible alkaloids thus do not entail chloroplast/cytosol separation of AS isoenzymes and yet might involve differential feedback regulation of pathway-specific AS alpha subunits. The two AS alpha isoenzymes of R. graveolens were expressed as glutathione S-transferase fusion proteins in Escherichia coli deletion mutants defective in AS activity and were purified to homogeneity. Differential sensitivity of the transformed E. coli strains toward 5-methyltryptophan, a false-feedback inhibitor of AS, was demonstrated. Characterization of affinity-purified AS alpha isoenzymes revealed that the noninducible AS alpha 2 of R. graveolens is strongly feedback inhibited by 10 microns tryptophan. In contrast, the elicitor-inducible AS alpha 1 isoenzyme is only slightly affected even by tryptophan concentrations 10-fold higher than those observed in planta. These results are consistent with the hypothesis that chorismate flux into biosynthesis of tryptophan and defense-related alkaloid biosynthesis in R. graveolens is regulated at the site of AS alpha isoenzymes at both genetic and enzymatic levels.     

Identification of amino acid residues involved in feedback regulation of the anthranilate synthase complex from Salmonella typhimurium. Evidence for an amino-terminal regulatory site

The anthranilate synthase-phosphoribosyl transferase complex, a heterotetrameric enzyme made up of the TrpE and TrpD polypeptides, catalyzes three reactions comprising the first two steps of tryptophan biosynthesis in Salmonella typhimurium. All three activities of the complex are subject to feedback inhibition by tryptophan, which results from allosteric effects associated with the binding of one molecule of inhibitor to each of the TrpE subunits of the complex. Random in vitro chemical mutagenesis of the trpE gene was used to generate a collection of mutant forms of the complex which displayed varying degrees of resistance to feedback inhibition. Single amino acid substitutions, identified by DNA sequencing, were found at 14 different residues within the TrpE polypeptide. The residues were distributed throughout TrpE, but those that appeared to be most critical for regulation were found in two clusters, one at the extreme amino-terminal end, including residues Glu-39, Ser-40, and Ala-41, and the other in the middle of the polypeptide, including residues Asn-288, Pro-289, Met-293, Phe-294, and Gly-305. Kinetic and binding studies of the purified mutant complexes demonstrated that 9 of the 14 had a marked decrease in affinity for tryptophan with little or no change in substrate affinity or catalytic capacity. The remaining five enzymes exhibited more subtle changes, having small decreases in inhibitor affinity coupled with small increases in substrate affinity. Mutant enzymes that were not totally feed-back-resistant had a decreased kinetic response to tryptophan binding. All enzymes exhibited alterations in tryptophan-induced conformational changes as monitored by dye-ligand chromatography.     

Tryptophan Synthetic Pathway and Its Regulation in Chromobacterium violaceum

Extracts of Chromobacterium violaceum catalyzed all of the reactions involved in synthesizing tryptophan from chorismic acid. Tryptophan auxotrophs which had lost any of these activities did not produce the characteristic purple pigment, violacein, when grown on a medium in which tryptophan was limiting. Gel filtration of extracts allowed us to estimate molecular weights for the tryptophan enzymes. All of the enzymes appeared to have molecular weights below 100,000. No enzymes were observed to occur in aggregates. The specific activities of the enzymes of the tryptophan pathway did not change when mutants were grown under conditions of limiting or excess tryptophan. The first enzyme in the pathway, anthranilate synthetase, was subject to feedback control by the end product, tryptophan. Tryptophan acted as a noncompetitive inhibitor with respect to glutamine, one of the substrates for anthranilate synthetase, and as a competitive inhibitor of the reaction when chorismate, the other substrate, was varied. The nonlinearity observed in the Lineweaver-Burk plot in the latter case suggests that there may be more than one chorismate-binding site on anthranilate synthetase.     

Purification and characterization of anthranilate synthase component I (TrpE) from Mycobacterium tuberculosis H37Rv

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis is the main reason why tuberculosis (TB) continues to be a major health problem worldwide. It is urgent to discover novel anti-mycobacterial agents based on new drug targets for the treatment of TB, especially MDR-TB. Tryptophan biosynthetic pathway, which is essential for the survival of M. tuberculosis and meanwhile absent in mammals, provides potential anti-TB drug targets. One of the promising drug targets in this pathway is anthranilate synthase component I (TrpE), whose role is to catalyze the conversion of chorismate to anthranilate using ammonia as amino source. In order to get a deep understanding of TrpE, a study on purification and characteristic identification of TrpE is required. In this work, the putative trpE gene of M. tuberculosis H37Rv was expressed as a fusion protein with a 6x His-tag on the N-terminal (His-TrpE) in Escherichia coli. The recombinant TrpE protein was successfully purified and then its enzymatic characteristics were analyzed. The native TrpE without His-tag was obtained by removal of the N-terminal fusion partner of His-TrpE using enterokinase. It was found that N-terminal fusion partner had little influence on TrpE catalytic activity. In addition, the key residues related to enzyme catalytic activity and that involved in l-tryptophan inhibition were predicted in the structure of M. tuberculosis H37Rv TrpE. These results would be beneficial to the designing of novel anti-TB drugs with high potency and selectivity.     

Modification of Serratia marcescens anthranilate synthase with pyridoxal 5′-phosphate

The glutamine-dependent activity of Serratia marcescens anthranilate synthase was inactivated by pyridoxal 5′-phosphate and sodium cyanide. The reaction was specific in that the ammonia-dependent activity of the enzyme was unaffected. The inactivation was stable to dilution or dialysis but was reversed by dithiothreitol. The enzyme contains dissimilar subunits designated anthranilate synthase components I (AS I) and II (AS II). Incorporation of [¹⁴C]NaCN demonstrates that modification was limited to one to two residues per AS I · AS II protomer. An active site cysteine is involved in the glutamine-dependent activity. Modification by pyridoxal 5′-phosphate and NaCN blocked affinity labeling of the active site cysteine by the glutamine analog 6-diazo-5-oxo-l-norleucine and reduced alkylation of the active site cysteine by iodoacetamide. These results suggest modification is at the glutamine active site. Initial modification by iodoacetamide did not prevent pyridoxal 5′-phosphate-dependent incorporation of ¹⁴CN showing that the pyridoxal 5′-phosphate modification did not involve the essential cysteinyl residue. These results suggest that modification of a lysyl residue in the glutamine active site of anthranilate synthase reduces the reactivity of the essential cysteinyl residue resulting in the loss of the amidotransferase activity.     

Studies on the subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

Both uncomplexed subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium have an absolute requirement for divalent metal ions which can be satisfied by Mg²⁺, Mn²⁺, or Co²⁺. The metal ion kinetics for uncomplexed anthranilate synthetase give biphasic double-reciprocal plots and higher apparent K_m values than those for anthranilate synthetase in the enzyme complex. In contrast, the apparent K_m values for phosphoribosyltransferase are the same whether the enzyme is uncomplexed or complexed with anthranilate synthetase. This suggests that the metal ion sites on anthranilate synthetase, but not those on phosphoribosyltransferase, are altered upon formation of the enzyme complex. These results and the results of studies reported by others, suggest that complex formation between anthranilate synthetase and phosphoribosyltransferase leads to marked alterations at the active site of the former, but not the latter enzyme. Uncomplexed anthranilate synthetase can be stoichiometrically labeled with Co(III) under conditions which lead to inactivation of 75% of its activity. A comparison of the effects of anthranilate and tryptophan on phosphoribosyltransferase activity in the uncomplexed and complexed forms shows that anthranilate, but not tryptophan, inhibits the uncomplexed enzyme. The complexed phosphoribosyltransferase shows substrate inhibition by anthranilate binding to the phosphoribosyltransferase subunits. In contrast, in a tryptophan-hypersensitive variant complex, anthranilate inhibits phosphoribosyltransferase activity by acting on the anthranilate synthetase subunits. The data are interpreted to mean that there are two classes of binding sites for anthranilate, one on each type of subunit, which may participate in the regulation of anthranilate synthetase and phosphoribosyltransferase under different conditions.     

Molecular basis of alpha-methyltryptophan resistance in amt-1, a mutant of Arabidopsis thaliana with altered tryptophan metabolism.

A mutant of Arabidopsis thaliana, amt-1, was previously selected for resistance to growth inhibition by the tryptophan analog alpha-methyltryptophan. This mutant had elevated tryptophan levels and exhibited higher anthranilate synthase (AS) activity that showed increased resistance to feedback inhibition by tryptophan. In this study, extracts of the mutant callus exhibited higher AS activity than wild-type callus when assayed with either glutamine or ammonium sulfate as amino donor, thus suggesting that elevated AS activity in the mutant was due to an alteration in the alpha subunit of the enzyme. The mutant also showed cross-resistance to 5-methylanthranilate and 6-methylanthranilate and mapped to chromosome V at or close to ASA1 (a gene encoding the AS alpha subunit). ASA1 mRNA and protein levels were similar in mutant and wild-type leaf extracts. Levels of ASA1 mRNA and protein were also similar in callus cultures of mutant and wild type, although the levels in callus were higher than in leaf tissue. Sequencing of the ASA1 gene from amt-1 revealed a G to A transition relative to the wild-type gene that would result in the substitution of an asparagine residue in place of aspartic acid at position 341 in the predicted amino acid sequence of the ASA1 protein. The mutant allele in strain amt-1 has been renamed trp5-1.     

Anthranilate synthase without an LLES motif from a hyperthermophilic archaeon is inhibited by tryptophan

Tk-trpE and Tk-trpG, the genes that encode the two subunits of anthranilate synthase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, have been expressed independently in Escherichia coli. The anthranilate synthase complex (Tk-AS complex) was obtained by heat-treatment of the mixture of cell-free extracts containing each recombinant protein, Tk-TrpE (alpha subunit) and Tk-TrpG (beta subunit), at 85 degrees C for 10 min. Further purification of Tk-AS complex was carried out by anion-exchange chromatography followed by gel-filtration. Molecular mass estimations from gel-filtration chromatography indicated that Tk-AS complex was a heterodimer (alphabeta). The complex displayed both ammonia- and glutamine-dependent anthranilate synthase activities, and could not utilize asparagine as an ammonia donor. The optimal pH was pH 10.0 and the optimal temperature was 85 degrees C in both cases. Mg2+ was necessary for the anthranilate synthase activity. At 75 degrees C, the K(m) values of chorismate for ammonia- and glutamine-dependent activities were 13.8 and 3.4 microM, respectively. The K(m) value of Mg2+ was 20.5 microM. The K(m) values of glutamine and NH4Cl were 88 microM and 5.6 mM, respectively. Although Tk-TrpE displayed 47.6% similarity with TrpE of Salmonella typhimurium, conserved amino acid residues proven to be essential for inhibition of enzyme activity by L-tryptophan were not present in Tk-TrpE. Namely, residues corresponding to Glu39, Met293, and Cys465 in the enzyme from S. typhimurium were replaced by Arg28, Thr221, and Ala384 in Tk-TrpE. Nevertheless, significant inhibition by L-tryptophan was observed, with K(i) values of 5.25 and 74 microM for ammonia and glutamine-dependent activities, respectively. The inhibition was competitive with respect to chorismate. The results suggest that the amino acid residues involved in the feedback inhibition by L-tryptophan in the case of Tk-AS complex are distinct from previously reported anthranilate synthases.     

Control of Tryptophan Biosynthesis in Plant Tissue Cultures: Lack of Repression of Anthranilate and Tryptophan Synthetases by Tryptophan

Tobacco (cv. Xanthi and cv. Wisconsin 38), rice, carrot, tomato, and soybean tissue cultures were grown in liquid media containing L-tryptophan. The addition of tryptophan increased the cellular tryptophan levels greatly (12–2500 fold), but did not lower appreciably the levels of two tryptophan biosynthetic enzymes, anthranilate synthetase and tryptophan synthetase. However, the addition of 50 μM tryptophan to the crude enzyme extract completely inhibited the anthranilate synthetase activity while 1 mM tryptophan inhibited the tryptophan synthetase activity by only 10–20°/o. This information indicates that tryptophan biosynthesis is controlled by the feedback inhibition of anthranilate synthetase by tryptophan and not by repression of enzyme synthesis. All of the species had significant enzyme levels. Anthranilate synthetase activity could not be detected in extracts from cells grown on tryptophan unless the extracts were first passed through two G-25 Sephadex columns with a short 30 °C warming step in between, a procedure shown to remove an inhibitor of the enzyme.     

Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase

Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.     

Structure of the cooperative allosteric anthranilate synthase from Salmonella typhimurium

We have determined the X-ray crystal structure of the cooperative anthranilate synthase heterotetramer from Salmonella typhimurium at 1.9 A resolution with the allosteric inhibitor l-tryptophan bound to a regulatory site in the TrpE subunit. Tryptophan binding orders a loop that in turn stabilizes the inactive T state of the enzyme by restricting closure of the active site cleft. Comparison with the structure of the unliganded, noncooperative anthranilate synthase heterotetramer from Sulfolobus solfataricus shows that the two homologs have completely different quarternary structures, even though their functional dimer pairs are structurally similar, consistent with differences in the cooperative behavior of the enzymes. The structural model rationalizes mutational and biochemical studies of the enzyme and establishes the structural differences between cooperative and noncooperative anthranilate synthase homologs.     

Constitutively active glutaminase variants provide insights into the activation mechanism of anthranilate synthase

The glutamine amidotransferase (GATase) family comprises enzyme complexes which consist of glutaminase and synthase subunits that catalyze in a concerted reaction the incorporation of nitrogen within various metabolic pathways. An important feature of GATases is the strong stimulation of glutaminase activity by the associated synthase. To understand the mechanism of this tight activity regulation, we probed by site-directed mutagenesis four residues of the glutaminase subunit TrpG from anthranilate synthase that are located between the catalytic Cys-His-Glu triad and the synthase subunit TrpE. In order to minimize structural perturbations induced by the introduced exchanges, the amino acids from TrpG were substituted with the corresponding residues of the closely related glutaminase HisH from imidazole glycerol phosphate synthase. Steady-state kinetic characterization showed that, in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyzed glutamine in the absence of TrpE. A reaction assay performed with hydroxylamine as a stronger nucleophile replacing water and a filter assay with radiolabeled glutamine indicated that the formation of the thioester intermediate is the rate-limiting step of constitutive glutamine hydrolysis. Molecular dynamics simulations with wild-type TrpG and constitutively active TrpG variants suggest that the introduced amino acid exchanges result in a distance reduction between the active site Cys-His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain. We propose that native TrpG in the anthranilate synthase complex is activated by a similar mechanism.