Structure of subunit I of the anthranilate synthase complex of Mycolicibacterium smegmatis.

The tryptophan biosynthesis pathway, which does not exist in mammals, is highly conserved in Mycobacterium. Anthranilate synthase (AS) catalyzes the initial reactions in the tryptophan biosynthesis pathway in many microorganisms, catalyzing the conversion of glutamine and chorismate to form pyruvate and anthranilate. Here, the crystal structure of anthranilate synthase component I (AS I) from Mycolicibacterium smegmatis (MsTrpE) has been determined to 1.7 Å resolution. MsTrpE crystallizes in the space group P1 with two monomers in the asymmetric unit, which is consistent with the oligomeric state in solution as confirmed by analytical ultracentrifugation. Inspection of the active site shows that it is in the active form with a bound Mg2+ ion and a ligand that is modelled as benzoate. The position of benzoate mimics the position of the anthranilate product in the active site. The structure of MsTrpE will provide a starting point for the investigation of latent biotechnology and pharmaceutical applications of anthranilate synthase component I.

Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes.

The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS.

Structure and inhibition of subunit I of the anthranilate synthase complex of Mycobacterium tuberculosis and expression of the active complex.

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.

The fused anthranilate synthase from Streptomyces venezuelae functions as a monomer.

Recently, we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here, we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography, and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2 ± 0.2 µM and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant k cat of 0.57 ± 0.02 s(-1) at 30 °C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but non-cooperatively inhibited by tryptophan (K i = 11.1 ± 0.1 µM) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme's minimal functional unit as a single TrpE-TrpG pair.

Conversion of anthranilate synthase into isochorismate synthase: implications for the evolution of chorismate-utilizing enzymes.

Chorismate-utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as well as free-living and infectious microorganisms. Among these enzymes are the homologous primary metabolic anthranilate synthase (AS) and secondary metabolic isochorismate synthase (ICS). Both catalyze mechanistically related reactions by using ammonia and water as nucleophiles, respectively. We report that the nucleophile specificity of AS can be extended from ammonia to water by just two amino acid exchanges in a channel leading to the active site. The observed ICS/AS bifunctionality demonstrates that a secondary metabolic enzyme can readily evolve from a primary metabolic enzyme without requiring an initial gene duplication event. In a general sense, these findings add to our understanding how nature has used the structurally predetermined features of enzyme superfamilies to evolve new reactions.

Reprogramming the Specificity of a Protein Interface by Computational and Data-Driven Design.

The formation of specific protein complexes in a cell is a non-trivial problem given the co-existence of thousands of different polypeptide chains. A particularly difficult case are two glutamine amidotransferase complexes (anthranilate synthase [AS] and aminodeoxychorismate synthase [ADCS]), which are composed of homologous pairs of synthase and glutaminase subunits. We have attempted to identify discriminating interface residues of the glutaminase subunit TrpG from AS, which are responsible for its specific interaction with the synthase subunit TrpEx and prevent binding to the closely related synthase subunit PabB from ADCS. For this purpose, TrpG-specific interface residues were grafted into the glutaminase subunit PabA from ADCS by two different approaches, namely a computational and a data-driven one. Both approaches resulted in PabA variants that bound TrpEx with higher affinity than PabB. Hence, we have accomplished a reprogramming of protein-protein interaction specificity that provides insights into the evolutionary adaptation of protein interfaces.

Pushing structural information into the yeast interactome by high-throughput protein docking experiments.

The last several years have seen the consolidation of high-throughput proteomics initiatives to identify and characterize protein interactions and macromolecular complexes in model organisms. In particular, more that 10,000 high-confidence protein-protein interactions have been described between the roughly 6,000 proteins encoded in the budding yeast genome (Saccharomyces cerevisiae). However, unfortunately, high-resolution three-dimensional structures are only available for less than one hundred of these interacting pairs. Here, we expand this structural information on yeast protein interactions by running the first-ever high-throughput docking experiment with some of the best state-of-the-art methodologies, according to our benchmarks. To increase the coverage of the interaction space, we also explore the possibility of using homology models of varying quality in the docking experiments, instead of experimental structures, and assess how it would affect the global performance of the methods. In total, we have applied the docking procedure to 217 experimental structures and 1,023 homology models, providing putative structural models for over 3,000 protein-protein interactions in the yeast interactome. Finally, we analyze in detail the structural models obtained for the interaction between SAM1-anthranilate synthase complex and the MET30-RNA polymerase III to illustrate how our predictions can be straightforwardly used by the scientific community. The results of our experiment will be integrated into the general 3D-Repertoire pipeline, a European initiative to solve the structures of as many as possible protein complexes in yeast at the best possible resolution. All docking results are available at http://gatealoy.pcb.ub.es/HT_docking/.

Inhibition of chorismate-utilising enzymes by 2-amino-4-carboxypyridine and 4-carboxypyridone and 5-carboxypyridone analogues.

Several 2-amino-4-carboxypyridine, 4- and 5-carboxypyridone-based compounds were prepared and tested against three members of the chorismate-utilising enzyme family, anthranilate synthase, isochorismate synthase and salicylate synthase. Most compounds exhibited low micromolar inhibition of these three enzymes. The most potent inhibitor was a 4-carboxypyridone analogue bearing a lactate side chain on the pyridyl nitrogen which exhibited inhibition constants of 5, 91 and 54 muM against anthranilate synthase, isochorismate synthase and salicylate synthase respectively.

Conformational changes in ammonia-channeling glutamine amidotransferases.

Glutamine amidotransferases (GATs), which catalyze the synthesis of different aminated products, channel ammonia over 10-40 A from a glutamine substrate at the glutaminase site to an acceptor substrate at the synthase site. Ammonia production usually uses a cysteine-histidine-glutamate triad or a N-terminal cysteine residue. Crystal structures of several amidotransferase ligand complexes, mimicking intermediates along the catalytic cycle, have now been determined. In most cases, acceptor binding triggers glutaminase activation through domain-hinged movements and other conformational changes. Structural information shows how flexible loops of the synthase and glutaminase domains move to shield the two catalytic sites and anchor the substrates, and how the ammonia channel forms and opens or closes.

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.

Mutations in the tryptophan operon allow PurF-independent thiamine synthesis by altering flux in vivo.

Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.

Anthranilate synthase subunit organization in Chromobacterium violaceum.

Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).

Metabolic flux analysis in plants using dynamic labeling technique: application to tryptophan biosynthesis in cultured rice cells.

The concept and methodology of using dynamic labeling for the MFA of plant metabolic pathways are described, based on a case study to develop a method for the MFA of the tryptophan biosynthetic pathway in cultured rice cells. Dynamic labeling traces the change in the labeling level of a metabolite in a metabolic pathway after the application of a stable isotope-labeled compound. In this study, [1-(13)C] l-serine was fed as a labeling precursor and the labeling level of Trp was determined by using the LC-MS/MS. The value of metabolic flux is determined by fitting a model describing the labeling dynamics of the pathway to the observed labeling data. The biosynthetic flux of Trp in rice suspension cultured cell was determined to be 6.0+/-1.1 nmol (gFWh)(-1). It is also demonstrated that an approximately sixfold increase in the biosynthetic flux of Trp in transgenic rice cells expressing the feedback-insensitive version of anthranilate synthase alpha-subunit gene (OASA1D) resulted in a 45-fold increase in the level of Trp. In this article, the basic workflow for the experiment is introduced and the details of the actual experimental procedures are explained. Future perspectives are also discussed by referring recent advances in the dynamic labeling approach.

[Cloning, sequence analysis and expression of anthranilate synthetase gene in Corynebacterium pekinense].

Anthranilate synthetase (EC4.1.3.27;AS) genes from wild-type Corynebacterium pekinense AS1.299 and its mutant PD-67 were cloned and sequenced. Analysis of PCR fragments revealed that three ORFs existed, which corresponded to trpL, trpE and trpG gene, respectively. Six bases changes that resulted in the changes of five amino acids were found in the trpE structural gene of C. pekinense PD-67 and a single-base change that resulted in an amino acid substitution was found in the trpG structural gene of C. pekinense PD-67.A homology comparison revealed that C. pekinense AS1.299 was closely related to Corynebacterim glutamicum ATCC 13032 and Brevibacterium lactofermentum. An internal promoter was found in the upstream of the trpL gene from C. pekinense and it functioned in E. coli, but a single-base exchange (A to G) existed in the-35 box of PD-67. The trpEG genes from the wild-type strain and its mutant were expressed both in C. pekinense AS1.299 and PD-67, and the specific enzyme activities of transformed C. pekinense were much higher than that of the parental strains. The amplification of the activity of AS yielded 22.39% increase of L-tryptophan production, but the cell growth became slower than PD-67.

Uncoupling of an ammonia channel as a mechanism of allosteric inhibition in anthranilate synthase of Serratia marcescens: dynamic and graph theoretical analysis.

Anthranilate synthase (AS) is the first branch node enzyme that catalyzes the conversion of chorismate to anthranilate in the high energy-consuming tryptophan biosynthetic pathway in Serratia marcescens. AS, with an allosterically-bound inhibitor (tryptophan), shows complete inhibition in its catalytic function, but the inhibitor-bound structure is very similar to that of the substrate-bound AS. Even though the reaction mechanisms of several chorismate-utilizing enzymes are known, the unusual structure-function relationship in catalysis and allosteric inhibition of AS by tryptophan, with an insignificant change in structure, remains elusive. In the absence of structural variation, we use an integrated computational approach of coarse-grained protein contact networks, Gaussian network model, and atomistic Molecular Dynamics simulations of the substrate-bound and inhibitor-bound AS structures, and show the role of small but critical allosteric changes that induce complete inhibition of AS activity. We predict, through dynamic correlation studies, perturbation in crucial inter-subunit interactions between the two substrate-binding sites ("ammonia channel") and the allosteric inhibitor-binding site, and identify, through shortest path analysis, the non-active site residues participating in the communication pathways. We argue that such a regulatory mechanism (change in function without a significant change in the structure) for catalysis is useful for a branch point enzyme that has to undergo fast redistribution of fluxes according to different metabolic states of the organism. Being essential to the survival of microorganisms, including pathogenic ones, and absent in mammals, AS is a highly attractive drug target. Thus, the allosteric AS residues participating in catalysis identified in this study could be important for drugability.

Chorismate lyase: kinetics and engineering for stability.

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis.

Anthranilate synthase I (ASI) of Bacillus caldotenax, a thermophilic bacterium, was purified from a plasmid-bearing Escherichia coli and characterized. The molecular weight determination under native and denaturing conditions revealed that it was a monomeric enzyme of M(r) = 54,000. The N-terminal amino acid sequence is the same as expected from DNA sequence of trpE except that the N-terminal methionine is lacking. All four cysteines in the molecule could be titrated with 5,5'-dithiobis (2-nitrobenzoic acid) in more than 8 M urea. The purified enzyme retained its full enzymatic activity after being heated at 60 degrees C. Six mutated genes for the ASI with histidine in place of each conserved arginine, Arg321, Arg353, Arg358, Arg416, Arg429, and Arg452, were prepared by site-directed mutagenesis. All the mutated genes except one, the gene encoding an ASI mutant with histidine in place of Arg452 (R452H ASI) complemented an E. coli (trpE). The mutated ASIs were purified and compared with the wild type ASI. No distinctive differences in enzymatic properties were found between the wild type and the enzymatically active mutated ASIs. R452H ASI was enzymatically inactive, though its conformation seemed to be unchanged after the substitution based on CD spectra and the SH titration curve.

An aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica is attenuated and immunogenic in a mouse model of infection.

We have constructed an aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica, harbouring mutations in aroA and trpE to investigate the use of such a strain as a live-attenuated vaccine. B. bronchiseptica aroA trpE was unable to grow in minimal medium without aromatic supplementation. Compared to the parental wild-type strain, the mutant displayed significantly reduced abilities to invade and survive within the mouse macrophage-like cell line J774A.1 in vitro and in the murine respiratory tract following experimental intranasal infection. Mice vaccinated with B. bronchiseptica aroA trpE displayed significant dose-dependent increases in B. bronchiseptica-specific antibody responses, and exhibited increases in the number of B. bronchiseptica-reactive spleen cells in lymphoproliferation assays. Immunised animals were protected against lung colonisation after challenge with the wild-type parental strain. With such a broad host range displayed by B. bronchiseptica, the attenuated strain constructed in this study may not only be used for the prevention of B. bronchiseptica-associated disease, but also for the potential delivery of heterologous antigen.

Thermodynamics of reactions catalyzed by anthranilate synthase.

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq) + ammonia(aq) = anthranilate(aq) + pyruvate(aq) + H2O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq) + ammonia(aq) = ADIC(aq) + H2O(l) and ADIC(aq) = anthranilate(aq) + pyruvate(aq). The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively. Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79. Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate2-(aq) + NH4+(aq) = anthranilate-(aq) + pyruvate-(aq) + H+(aq) + H2O(l), delta rHmo = -(116.3 +/- 5.4) kJ mol-1. For the reaction: chorismate2-(aq) + NH4+(aq) = ADIC-(aq) + H2O(l), K = (20.3 +/- 4.5) and delta rHmo = (7.5 +/- 0.6) kJ mol-1. Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes delta rG'mo under approximately physiological conditions are given.

[Differential expression of recombinant hybrid proteins containing amino- and carboxy-terminal regions of the exterior large glycoprotein (gp46) of the human T-cell leukemia virus (HTLV-I) in Escherichia coli cells]. / Differentsial'naia ékspressiia rekombinantnykh gibridnykh belkov, soderzhashchikh amino- i karboksiterminal'nuiu oblasti naruzhnogo bol'shogo glikoproteida (gp46) virusa T-kletochnogo leikoza cheloveka (HTLV-I) b kletkakh Escherichia coli.

Plasmid clones capable of expressing a recombinant fusion proteins containing the anthranilate synthase of E. coli (TrpE) and different regions of gp46 HTLV-I were constructed on the basis of pATH-vectors. A high extent of TrpE-gp46 proteolytic degradation took place independently of the bacterial La-protease. Fusion proteins containing an N-terminal part of gp46 were more stable and could be purified in preparative quantities but were less antigenic. On the contrary, a TrpE-gp46 protein encoded by the TaqI-TaqI DNA fragment and containing only 35 C-terminal amino acids was still susceptible to degradation but possessed good serologic reactivity. Some of the recombinant proteins obtained can be useful for diagnostics and for preparing monoclonal or polyclonal antibodies.