Crystal structures of Yersinia enterocolitica salicylate synthase and its complex with the reaction products salicylate and pyruvate

The salicylate synthase, Irp9, from Yersinia enterocolitica is involved in the biosynthesis of the siderophore yersiniabactin. It is a bifunctional enzyme that forms salicylate and pyruvate from chorismate and water via the intermediate isochorismate. Here we report the first crystal structure of Irp9 and also of its complex with the reaction products salicylate and pyruvate at 1.85 A and 2.1 A resolution, respectively. Like other members of the chorismate-utilizing enzyme family, e.g. the TrpE subunit of anthranilate synthase and the PabB subunit of 4-amino-4-deoxychorismate synthase, Irp9 has a complex alpha/beta fold. The crystal structure of Irp9 contains one molecule each of phosphate and acetate derived from the crystallization buffer. The Irp9-products complex structure was obtained by soaking chorismate into Irp9, demonstrating that the enzyme is still catalytically active in the crystal. Both structures contain Mg(2+) in the active site. There is no evidence of the allosteric tryptophan binding site found in TrpE and PabB. Mutagenesis of Glu240, His321 and Tyr372 provided some insight into the mechanism of the two transformations catalyzed by Irp9. Knowledge of the structure of Irp9 will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake, which is directly related to the virulence of Yersinia.     

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.     

Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited

The isochorismate synthase from Pseudomonas aeruginosa (PchA) catalyzes the conversion of chorismate to isochorismate, which is subsequently converted by a second enzyme (PchB) to salicylate for incorporation into the salicylate-capped siderophore pyochelin. PchA is a member of the MST family of enzymes, which includes the structurally homologous isochorismate synthases from Escherichia coli (EntC and MenF) and salicylate synthases from Yersinia enterocolitica (Irp9) and Mycobacterium tuberculosis (MbtI). The latter enzymes generate isochorismate as an intermediate before generating salicylate and pyruvate. General acid–general base catalysis has been proposed for isochorismate synthesis in all five enzymes, but the residues required for the isomerization are a matter of debate, with both lysine221 and glutamate313 proposed as the general base (PchA numbering). This work includes a classical characterization of PchA with steady state kinetic analysis, solvent kinetic isotope effect analysis and by measuring the effect of viscosogens on catalysis. The results suggest that isochorismate production from chorismate by the MST enzymes is the result of general acid–general base catalysis with a lysine as the base and a glutamic acid as the acid, in reverse protonation states. Chemistry is determined to not be rate limiting, favoring the hypothesis of a conformational or binding step as the slow step.     

Salicylate biosynthesis: overexpression, purification, and characterization of Irp9, a bifunctional salicylate synthase from Yersinia enterocolitica

In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K(m) for chorismate of 4.2 microM and a k(cat) of 8 min(-1). The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1H NMR spectroscopy.     

Irp9, encoded by the high-pathogenicity island of Yersinia enterocolitica,is able to convert chorismate into salicylate,the precursor of the siderophore yersiniabactin

The Irp9 protein of Yersinia enterocolitica participates in the synthesis of salicylate, the precursor of the siderophore yersiniabactin. In Pseudomonas species, salicylate synthesis is mediated by two enzymes: isochorismate synthase and isochorismate pyruvate-lyase. Both enzymes are required for complementation of a Yersinia irp9 mutant. However, irp9 is not able to complement Escherichia coli entC for the production of enterobactin, which requires isochorismate as a precursor. These results suggest that Irp9 directly converts chorismate into salicylate.     

Crystal Structure of Escherichia coli Enterobactin-specific Isochorismate Synthase (EntC) Bound to its Reaction Product Isochorismate: Implications for the Enzyme Mechanism and Differential Activity of Chorismate-utilizing Enzymes

EntC, one of two isochorismate synthases in Escherichia coli, is specific to the biosynthesis of the siderophore enterobactin. Here, we report the crystal structure of EntC in complex with isochorismate and Mg²⁺at 2.3 Å resolution, the first structure of a chorismate-utilizing enzyme with a non-aromatic reaction product. EntC exhibits a complex α+β fold like the other chorismate-utilizing enzymes, such as salicylate synthase and anthranilate synthase. Comparison of active site structures allowed the identification of several residues, not discussed previously, that might be important for the isochorismate activity of the EntC. Although EntC, MenF and Irp9 all convert chorismate to isochorismate, only Irp9 subsequently exhibits isochorismate pyruvate lyase activity resulting in the formation of salicylate and pyruvate as the reaction products. With a view to understanding the roles of these amino acid residues in the conversion of chorismate to isochorismate and to obtaining clues about the pyruvate lyase activity of Irp9, several mutants of EntC were generated in which the selected residues in EntC were substituted for those of Irp9: these included A303T, L304A, F327Y, I346L and F359Q mutations. Biochemical analysis of these mutants indicated that the side chain of A303 in EntC may be crucial in the orientation of the carbonyl to allow formation of a hydrogen bond with isochorismate. Some mutations, such as L304A and F359Q, give rise to a loss of catalytic activity, whereas others, such as F327Y and I346L, show that subtle changes in the otherwise closely similar active sites influence activity. We did not find a combination of these residues that conferred pyruvate lyase activity.     

Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis

MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family.     

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.     

Pericyclic reactions catalyzed by chorismate-utilizing enzymes

One of the fundamental questions of enzymology is how catalytic power is derived. This review focuses on recent developments in the structure--function relationships of chorismate-utilizing enzymes involved in siderophore biosynthesis to provide insight into the biocatalysis of pericyclic reactions. Specifically, salicylate synthesis by the two-enzyme pathway in Pseudomonas aeruginosa is examined. The isochorismate-pyruvate lyase is discussed in the context of its homologues, the chorismate mutases, and the isochorismate synthase is compared to its homologues in the MST family (menaquinone, siderophore, or tryptophan biosynthesis) of enzymes. The tentative conclusion is that the activities observed cannot be reconciled by inspection of the active site participants alone. Instead, individual activities must arise from unique dynamic properties of each enzyme that are tuned to promote specific chemistries.     

Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis.

In Escherichia coli, isochorismate is a common precursor for the biosynthesis of the siderophore enterobactin and menaquinone (vitamin K2). Isochorismate is formed by the shikimate pathway from chorismate by the enzyme isochorismate synthase encoded by the entC gene. Since enterobactin is involved in the aerobic assimilation of iron, and menaquinone is involved in anaerobic electron transport, we investigated the regulation of entC by iron and oxygen. An operon fusion between entC with its associated regulatory region and lacZ+ was constructed and introduced into the chromosome in a single copy. Expression of entC-lacZ was found to be regulated by the concentration of iron both aerobically and anaerobically. An established entC::kan mutant deficient in enterobactin biosynthesis was found to grow normally and synthesize wild-type levels of menaquinone under anaerobic conditions in iron-sufficient media. These results led to the demonstration of an alternate isochorismate synthase specifically involved in menaquinone synthesis encoded by the menF gene. Consistent with these findings, the entC+ strains were found to synthesize enterobactin anaerobically under iron-deficient conditions while the ent mutants failed to do so.     

Mutational and phylogenetic analyses of the mycobacterial mbt gene cluster

The mycobactin siderophore system is present in many Mycobacterium species, including M. tuberculosis and other clinically relevant mycobacteria. This siderophore system is believed to be utilized by both pathogenic and nonpathogenic mycobacteria for iron acquisition in both in vivo and ex vivo iron-limiting environments, respectively. Several M. tuberculosis genes located in a so-called mbt gene cluster have been predicted to be required for the biosynthesis of the core scaffold of mycobactin based on sequence analysis. A systematic and controlled mutational analysis probing the hypothesized essential nature of each of these genes for mycobactin production has been lacking. The degree of conservation of mbt gene cluster orthologs remains to be investigated as well. In this study, we sought to conclusively establish whether each of nine mbt genes was required for mycobactin production and to examine the conservation of gene clusters orthologous to the M. tuberculosis mbt gene cluster in other bacteria. We report a systematic mutational analysis of the mbt gene cluster ortholog found in Mycobacterium smegmatis. This mutational analysis demonstrates that eight of the nine mbt genes investigated are essential for mycobactin production. Our genome mining and phylogenetic analyses reveal the presence of orthologous mbt gene clusters in several bacterial species. These gene clusters display significant organizational differences originating from an intricate evolutionary path that might have included horizontal gene transfers. Altogether, the findings reported herein advance our understanding of the genetic requirements for the biosynthesis of an important mycobacterial secondary metabolite with relevance to virulence.     

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli.

The first committed step in the biosynthesis of menaquinone (vitamin K2) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene. This isochorismate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in enterobactin biosynthesis. MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity. The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 48 kDa. Thus, the enzyme is a homodimer. The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37 degrees C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg2+. The enzyme was found to have a Km of 195 +/- 23 microM and a k(cat) of 80 min(-1). In the presence of 30 mM beta-mercaptoethanol (BME), the k(cat) increased to 176 min(-1). The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold. Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity. Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted in complete protection of the enzyme from inactivation.     

Lysine 190 is the catalytic base in MenF, the menaquinone-specific isochorismate synthase from Escherichia coli: implications for an enzyme family

Menaquinone biosynthesis is initiated by the conversion of chorismate to isochorismate, a reaction that is catalyzed by the menaquinone-specific isochorismate synthase, MenF. The catalytic mechanism of MenF has been probed using a combination of structural and biochemical studies, including the 2.5 A structure of the enzyme, and Lys190 has been identified as the base that activates water for nucleophilic attack at the chorismate C2 carbon. MenF is a member of a larger family of Mg2+ dependent chorismate binding enzymes catalyzing distinct chorismate transformations. The studies reported here extend the mechanism recently proposed for this enzyme family by He et al.: He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378-85.     

Overexpression, purification, and characterization of isochorismate synthase (EntC), the first enzyme involved in the biosynthesis of enterobactin from chorismate

Isochorismate synthase (EC 5.4.99.6), the entC gene product of Escherichia coli, catalyzes the conversion of chorismate to isochorismate, the first step in the biosynthesis of the powerful iron-chelating agent enterobactin. A sequence-specific deletion method has been used to construct an EntC overproducer, which allows for the purification and characterization of the E. coli isochorismate synthase for the first time. The N-terminal sequence and the subunit molecular weight (43,000) of the polypeptide derived from SDS-polyacrylamide gel electrophoresis agree with those deduced from DNA sequence data. The enzyme is an active monomer with a native molecular weight of 42,000. It was shown that EntC alone is fully capable of catalyzing the interconversion of chorismate and isochorismate in both directions and the associated activity is not affected by EntA of the same biosynthetic pathway as has recently been speculated [Elkins, M. F., & Earhart, C. F. (1988) FEMS Microbiol. Lett. 56, 35; Liu, J., Duncan, K., & Walsh, C.T. (1989) J. Bacteriol. 171, 791; Ozenberger, B. A., Brickman, T.J., & McIntosh, M. A. (1989) J. Bacteriol. 171, 775]. The kinetic constants were determined with Km = 14 microM and kcat = 173 min-1 for chorismate in the forward direction and Km = 5 microM and kcat = 108 min-1 for isochorismate in the backward direction. The equilibrium constant for the reaction derived from the kinetic data is 0.56 with the equilibrium lying toward the side of chorismate, corresponding to a free energy difference of 0.36 kcal/mol between chorismate and isochorismate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase–prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.     

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.     

Mycobactin and the competition for iron between Mycobacterium neoaurum and M. vaccae

Two closely related species of mycobacteria, Mycobacterium vaccae and M. neoaurum, were grown under conditions of iron-deficiency (0.02-0.05 microgram Fe ml-1) and iron-sufficiency (2-4 micrograms Fe ml-1) in a simple glycerol/asparagine medium. The strain of M. vaccae used was a nonmycobactin producer whereas M. neoaurum synthesized between 6-8% of its cell biomass as the lipid-soluble siderophore when grown under iron-limitation. The role of mycobactin for iron-acquisition was examined using both pure and mixed cultures, with cell viability determined following growth at various iron concentrations. M. neoaurum, the mycobactin producer, outgrew M. vaccae when iron was readily available. When grown under conditions where iron was limiting, M. neoaurum showed a decline in viable cell number compared with its competitor, highlighting its increased requirement for the metal. Some recovery was observed following mycobactin biosynthesis, this being greatly enhanced by the addition of an iron supplement to the growing cells. Mycobactin biosynthesis allowed M. neoaurum to rapidly acquire any additional iron presented to the bacteria when growing under iron-limitation. However, M. vaccae did not synthesize the lipid-soluble siderophore with its iron-requirement satisfied by production of extracellular exochelin.     

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.