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.     

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.     

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.     

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.     

Exploration of swapping enzymatic function between two proteins: A simulation study of chorismate mutase and isochorismate pyruvate lyase

The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non‐native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues.     

Purification and Characterization of Chorismate Synthase from Euglena gracilis: Comparison with Chorismate Synthases of Plant and Microbial Origin

Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia coli), and a fungus (Neurospora crassa). These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli.     

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.     

A unique reaction in a common pathway: mechanism and function of chorismate synthase in the shikimate pathway

Chorismate synthase, the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi and plants. The enzyme has an absolute requirement for reduced FMN as a cofactor, although the 1,4-anti elimination of phosphate and the C(6proR)-hydrogen does not involve a net redox change. The role of the reduced FMN in catalysis has long been elusive. However, recent detailed kinetic and bioorganic approaches have fundamentally advanced our understanding of the mechanism of action, suggesting an initial electron transfer from tightly bound reduced flavin to the substrate, a process which results in C—O bond cleavage. Studies on chorismate synthases from bacteria, fungi and plants revealed that in these organisms the reduced FMN cofactor is made available in different ways to chorismate synthase: chorismate synthases in fungi – in contrast to those in bacteria and plants – carry a second enzymatic activity which enables them to reduce FMN at the expense of NADPH. Yet, as shown by the analysis of the corresponding genes, all chorismate synthases are derived from a common ancestor. However, several issues revolving around the origin of reduced FMN, as well as the possible regulation of the enzyme activity by means of the availability of reduced FMN, remain poorly understood. This review summarizes recent developments in the biochemical and genetic arena and identifies future aims in this field. Received: 22 June 1998 / Accepted: 7 August 1998     

Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg^2 + dependent and catalyzes a similar S_N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give K_chorismate = 20 ± 4 μM, K_Mg^2 + = 294 ± 22 μM, K_L-gln = 11 ± 1 mM, and k_cat = 2.2 ± 0.2 s^− 1 for a random kinetic mechanism. PhzE can use NH₄⁺ as an alternative nucleophile, while Co^2 + and Mn^2 + are alternative divalent metals.     

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.     

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.     

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

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

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK a

Several small ribozymes employ general acid–base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK_a values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK_a values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid–base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid–base catalysis indicate that k₁, the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pK_a. The inference that k₁ is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK_a is that of the general acid and the higher pK_a is that of the general base.     

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Pseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec(-1). In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (k(Ψ)) by TruB was determined to be 0.5 sec(-1). This rate constant is similar to the k(cat) obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec(-1), respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism.     

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.     

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)     

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.