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Nature of allosteric inhibition in glutamate racemase: discovery and characterization of a cryptic inhibitory pocket using atomistic MD simulations and pKa calculations.

ABSTRACT: Enzyme inhibition via allostery, in which the ligand binds remotely from the active site, is a poorly understood phenomenon and represents a significant challenge to structure-based drug design. Dipicolinic acid (DPA), a major component of Bacillus spores, is shown to inhibit glutamate racemase from Bacillus anthracis , a monosubstrate/monoproduct enzyme, in a novel allosteric fashion. Glutamate racemase has long been considered an important drug target for its integral role in bacterial cell wall synthesis. The DPA binding mode was predicted via multiple docking studies and validated via site-directed mutagenesis at the binding locus, while the mechanism of inhibition was elucidated with a combination of Blue Native polyacrylamide gel electrophoresis, molecular dynamics simulations, and free energy and pK(a) calculations. Inhibition by DPA not only reveals a novel cryptic binding site but also represents a form of allosteric regulation that exploits the interplay between enzyme conformational changes, fluctuations in the pK(a) values of buried residues and catalysis. The potential for future drug development is discussed.

SUBMITTER: Whalen KL

PROVIDER: S-EPMC3072873 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Nature of allosteric inhibition in glutamate racemase: discovery and characterization of a cryptic inhibitory pocket using atomistic MD simulations and pKa calculations.

Whalen Katie L KL Tussey Kenneth B KB Blanke Steven R SR Spies M Ashley MA

The journal of physical chemistry. B 20110311 13

Enzyme inhibition via allostery, in which the ligand binds remotely from the active site, is a poorly understood phenomenon and represents a significant challenge to structure-based drug design. Dipicolinic acid (DPA), a major component of Bacillus spores, is shown to inhibit glutamate racemase from Bacillus anthracis , a monosubstrate/monoproduct enzyme, in a novel allosteric fashion. Glutamate racemase has long been considered an important drug target for its integral role in bacterial cell wa ...[more]

PMID: 21395329

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Project description:Glutamate racemase (GR) catalyzes the cofactor independent stereoinversion of l- to d-glutamate for biosynthesis of bacterial cell walls. Because of its essential nature, this enzyme is under intense scrutiny as a drug target for the design of novel antimicrobial agents. However, the flexibility of the enzyme has made inhibitor design challenging. Previous steered molecular dynamics (MD), docking, and experimental studies have suggested that the enzyme forms highly varied complexes with different competitive inhibitor scaffolds. The current study employs a mutant orthogonal tRNA/aminoacyl-tRNA synthetase pair to genetically encode a non-natural fluorescent amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine (7HC), into a region (Tyr53) remote from the active site (previously identified by MD studies as undergoing ligand-associated changes) to generate an active mutant enzyme (GRY53/7HC). The GRY53/7HC enzyme is an active racemase, which permitted us to examine the nature of these idiosyncratic ligand-associated phenomena. One type of competitive inhibitor resulted in a dose-dependent quenching of the fluorescence of GRY53/7HC, while another type of competitive inhibitor resulted in a dose-dependent increase in fluorescence of GRY53/7HC. In order to investigate the environmental changes of the 7HC ring system that are distinctly associated with each of the GRY53/7HC-ligand complexes, and thus the source of the disparate quenching phenomena, a parallel computational study is described, which includes essential dynamics, ensemble docking and MD simulations of the relevant GRY53/7HC-ligand complexes. The changes in the solvent exposure of the 7HC ring system due to ligand-associated GR changes are consistent with the experimentally observed quenching phenomena. This study describes an approach for rationally predicting global protein allostery resulting from enzyme ligation to distinctive inhibitor scaffolds. The implications for fragment-based drug discovery and high throughput screening are discussed.

Project description:Project description: In this study, we have characterized with distinct biochemical assays the activity of a predicted glutamate racemase from an uncultivated bacterium (Candidatus Saccharimonas aalborgenesis), classified taxonomically within the Candidate Phyla Radiation (CPR). This racemase was produced in a Salmonella auxotrophic mutant defective in its endogenous glutamate racemase. Despite the low identity of the predicated racemase from this uncultivated bacterium compared to that of Salmonella (MurI), ~32.0%, it exhibited high specificity for glutamate as substrate in in vitro racemization assays among the rest of canonical amino acids. Moreover, the expression of this novel racemase complemented the D-Glu auxotrophy of the host bacterium used as chassis (Salmonella). Besides these physiological analyses, the study also characterized the composition and structure of the peptidoglycan produced by bacteria expressing each of the two glutamate racemases, exogenous and endogenous. Data processing protocol: Three biological replicates of two Salmonella strains expressing either the predicted glutamate racemase from the uncultivated bacterium Candidatus Saccharimonas aalborgenesis (Delta-murI-CPR, R1-R3) or the endogenous glutamate racemase (MurI) (Delta-murI-SAL, R1-R3) were grown in the nutrient rich medium LB to reach the exponential phase. At this time, bacteria were collected by centrifugation, washed and lysed by boiling in a 4% SDS-containing buffer. From this material, peptidoglycan was purified by standard protocol. Briefly, after high-speed centrifugation, the pellet containing peptidoglycan as the only SDS-insoluble material was subjected to successive enzymatic treatments with alpha-amylase, pronase (as protease) and, muramidase to cleave the glycan chains and to yield individual uncross-linked and cross-linked muropeptides. The muropeptide mixtures were analysed by LC-MS/MS to obtain a list of parental masses representing the complexity of the sample, i.e. the diversity of co-existing muropeptide classes. The parental masses were assigned as "muropeptides" when a mass of 204.3, corresponding to the N-acetyl-glucosamine molecule, was detected in the fragmentation spectra. The complete lists of parental masses were examined using FreeStyle (Thermo) software and compared among samples (biological replicas and control-problem pairs) by statistical parameters using XCMS software.

Dataset Information

Nature of allosteric inhibition in glutamate racemase: discovery and characterization of a cryptic inhibitory pocket using atomistic MD simulations and pKa calculations.

Publications

Nature of allosteric inhibition in glutamate racemase: discovery and characterization of a cryptic inhibitory pocket using atomistic MD simulations and pKa calculations.

Similar Datasets

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