title: Determinants of catalytic power and ligand binding in glutamate racemase.
abstract: Glutamate racemases (EC 5.1.1.3) catalyze the cofactor-independent stereoinversion of D- and L-glutamate and are important for viability in several gram-negative and -positive bacteria. As the only enzyme involved in the stereoinversion of L- to D-glutamate for peptidoglycan biosynthesis, glutamate racemase is an attractive target for the design of antibacterial agents. However, the development of competitive tight-binding inhibitors has been problematic and highly species specific. Despite a number of recent crystal structures of cofactor-independent epimerases and racemases, cocrystallized with substrates or substrate analogues, the source of these enzymes' catalytic power and their ability to acidify the C alpha of amino acids remains unknown. The present integrated computational and experimental study focuses on the glutamate racemase from Bacillus subtilis (RacE). A particular focus is placed on the interaction of the glutamate carbanion intermediate with RacE. Results suggest that the reactive form of the RacE-glutamate carbanion complex, vis-à-vis proton abstraction from C alpha, is significantly different than the RacE-D-glutamate complex on the basis of the crystal structure and possesses dramatically stronger enzyme-ligand interaction energy. In silico and experimental site-directed mutagenesis indicates that the strength of the RacE-glutamate carbanion interaction energy is highly distributed among numerous electrostatic interactions in the active site, rather than being dominated by strong hydrogen bonds. Results from this study are important for laying the groundwork for discovery and design of high-affinity ligands to this class of cofactor-independent racemases.
authors.affiliation: Department of Biochemistry, Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801, USA. aspies@life.uiuc.edu
authors.forename: M Ashley
authors.initials: MA
authors.lastname: Spies
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Reese
authors.forename: Dylan
authors.initials: D
authors.lastname: Dodd
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Pankow
authors.forename: Steven R
authors.initials: SR
authors.lastname: Blanke
authors.forename: Jerome
authors.initials: J
authors.lastname: Baudry
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D018698:Glutamic Acid; D000591:Amino Acid Isomerases; C062292:glutamate racemase