Unraveling the Structure and Mechanism of the MST(ery) Enzymes.
ABSTRACT: The menaquinone, siderophore, and tryptophan (MST) enzymes transform chorismate to generate precursor molecules for the biosynthetic pathways defined in their name. Kinetic data, both steady-state and transient-state, and X-ray crystal structures indicate that these enzymes are highly conserved both in mechanism and in structure. Because these enzymes are found in pathogens but not in humans, there is considerable interest in these enzymes as drug design targets. While great progress has been made in defining enzyme structure and mechanism, inhibitor design has lagged behind. This review provides a detailed description of the evidence that begins to unravel the mystery of how the MST enzymes work, and how that information has been used in inhibitor design.
Project description:The shikimate pathway of bacteria, fungi, and plants generates chorismate, which is drawn into biosynthetic pathways that form aromatic amino acids and other important metabolites, including folates, menaquinone, and siderophores. Many of the pathways initiated at this branch point transform chorismate using an MST enzyme. The MST enzymes (menaquinone, siderophore, and tryptophan biosynthetic enzymes) are structurally homologous and magnesium-dependent, and all perform similar chemical permutations to chorismate by nucleophilic addition (hydroxyl or amine) at the 2-position of the ring, inducing displacement of the 4-hydroxyl. The isomerase enzymes release isochorismate or aminodeoxychorismate as the product, while the synthase enzymes also have lyase activity that displaces pyruvate to form either salicylate or anthranilate. This has led to the hypothesis that the isomerase and lyase activities performed by the MST enzymes are functionally conserved. Here we have developed tailored pre-steady-state approaches to establish the kinetic mechanisms of the isochorismate and salicylate synthase enzymes of siderophore biosynthesis. Our data are centered on the role of magnesium ions, which inhibit the isochorismate synthase enzymes but not the salicylate synthase enzymes. Prior structural data have suggested that binding of the metal ion occludes access or egress of substrates. Our kinetic data indicate that for the production of isochorismate, a high magnesium ion concentration suppresses the rate of release of product, accounting for the observed inhibition and establishing the basis of the ordered-addition kinetic mechanism. Moreover, we show that isochorismate is channeled through the synthase reaction as an intermediate that is retained in the active site by the magnesium ion. Indeed, the lyase-active enzyme has 3 orders of magnitude higher affinity for the isochorismate complex relative to the chorismate complex. Apparent negative-feedback inhibition by ferrous ions is documented at nanomolar concentrations, which is a potentially physiologically relevant mode of regulation for siderophore biosynthesis in vivo.
Project description: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.
Project description: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.
Project description:PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. The PhzE monomer contains both a chorismate-converting menaquinone, siderophore, tryptophan biosynthesis (MST) and a type 1 glutamine amidotransferase (GATase1) domain connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligand-free open and ligand-bound closed conformation at 2.9 and 2.1 Å resolution, respectively. PhzE arranges in an intertwined dimer such that the GATase1 domain of one chain provides NH(3) to the MST domain of the other. This quaternary structure was confirmed by small angle x-ray scattering. Binding of chorismic acid, which was found converted to benzoate and pyruvate in the MST active centers of the closed form, leads to structural rearrangements that establish an ammonia transport channel approximately 25 Å in length within each of the two MST/GATase1 functional units of the dimer. The assignment of PhzE as an ADIC synthase was confirmed by mass spectrometric analysis of the product, which was also visualized at 1.9 Å resolution by trapping in crystals of an inactive mutant of PhzD, an isochorismatase that catalyzes the subsequent step in phenazine biosynthesis. Unlike in some of the related anthranilate synthases, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1 active center was identified as zinc, and it was demonstrated that Zn(2+), Mn(2+), and Ni(2+) reduce the activity of PhzE.
Project description:The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the siderophore bacillibactin by this pathogenic bacterium. The structure of the selenomethionine-substituted protein was determined to 2.4?Å resolution using single-wavelength anomalous diffraction. B. anthracis DhbC bears the strongest resemblance to the Escherichia coli isochorismate synthase EntC, which is involved in the biosynthesis of another siderophore, namely enterobactin. Both proteins adopt the characteristic fold of other chorismate-utilizing enzymes, which are involved in the biosynthesis of various products, including siderophores, menaquinone and tryptophan. The conservation of the active-site residues, as well as their spatial arrangement, suggests that these enzymes share a common Mg(2+)-dependent catalytic mechanism.
Project description: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.
Project description: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.
Project description:Reactivation of the telomerase reverse transcriptase subunit, TERT, is linked to tumourigenesis due to well-documented telomere-dependent and independent functions. The aim of this study was to investigate the effect of the telomerase inhibitor, MST-312, on TERT functions, focusing in particular, on its effects on MYC stabilty and MYC-regulated pathways, in order to assess its potential as a therapeutic agent. We demonstrate that MST-312 reduces MYC levels in cancer cells, leading to reduced MYC levels on chromatin, and subsequently affecting the MYC-regulated transcriptional program. As a result, MST-312 treatment increases the survival of lymphoma-bearing mice. Mechanistically, MST-312 affects the conformation of TERT, leading to TERT/Terc dissociation, and the subsequent loss of both its telomere-dependent and independent functions. Based on the presented data, we conclude that MST-312 treatment is a promising therapeutic strategy, in particular, in MYC-driven tumorus. Overall design: Total RNA was extracted from P493 cells treated with MST-312 and TET for 48 h, then released into fresh TET-free media with MST-312 for 0, 4, 8 and 24 hours.
Project description:Reactivation of the telomerase reverse transcriptase subunit, TERT, is linked to tumourigenesis due to well-documented telomere-dependent and independent functions. The aim of this study was to investigate the effect of the telomerase inhibitor, MST-312, on TERT functions, focusing in particular, on its effects on MYC stabilty and MYC-regulated pathways, in order to assess its potential as a therapeutic agent. We demonstrate that MST-312 reduces MYC levels in cancer cells, leading to reduced MYC levels on chromatin, and subsequently affecting the MYC-regulated transcriptional program. As a result, MST-312 treatment increases the survival of lymphoma-bearing mice. Mechanistically, MST-312 affects the conformation of TERT, leading to TERT/Terc dissociation, and the subsequent loss of both its telomere-dependent and independent functions. Based on the presented data, we conclude that MST-312 treatment is a promising therapeutic strategy, in particular, in MYC-driven tumorus. Overall design: ChIP was performed using anti-MYC (sc-764) in P493 cells and treated with MST-312.
Project description:The electron carrier menaquinone is one of many important bacterial metabolites that are derived from the key intermediate chorismic acid. MenF, the first enzyme in the menaquinone pathway, catalyzes the isomerization of chorismate to isochorismate. Here, an improved structure of MenF in a new crystal form is presented. The structure, solved at 2.0 angstroms resolution in complex with magnesium, reveals a well defined closed active site. Existing evidence suggests that the mechanism of the reaction catalyzed by MenF involves nucleophilic attack of a water molecule on the chorismate ring. The structure reveals a well defined water molecule located in an appropriate position for activation by Lys190 and attack on the substrate.