title: Understanding the determinants of substrate specificity in IMP family metallo-β-lactamases: the importance of residue 262.
abstract: In Gram-negative bacteria, resistance to β-lactam antibacterials is largely due to β-lactamases and is a growing public health threat. One of the most concerning β-lactamases to evolve in bacteria are the Class B enzymes, the metallo-β-lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel β-lactams and inhibitors. Two MBLs, the wild type IMP-1 with serine at position 262, and an engineered variant with valine at the same position (IMP-1-S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP-1-S262T) on the substrate spectrum. Here, we explore MBL sequence-structure-activity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP-1. Using site-directed mutagenesis, threonine was introduced at position 262, and the IMP-1-S262T enzyme, as well as the other two enzymes IMP-1 and IMP-1-S262V, were purified and kinetic constants were determined against a range of β-lactam antibacterials. Catalytic efficiencies (kcat /KM ) obtained with IMP-1-S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP-1 and IMP-1-S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in β-lactam turnover and support this approach to predict activities of certain novel MBL variants.
authors.affiliation: Biological Sciences Department, College of Science, California State Polytechnic University, Pomona, California.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Pegg
authors.forename: Eleanor M
authors.initials: EM
authors.lastname: Liu
authors.forename: Alex C
authors.initials: AC
authors.lastname: George
authors.forename: Alecander E
authors.initials: AE
authors.lastname: LaCuran
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.forename: Peter
authors.initials: P
authors.lastname: Oelschlaeger
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D002512:Cephalothin / Q000494:pharmacology; D014644:Genetic Variation; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism; D014633:Valine / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013912:Threonine; D012694:Serine; D001618:beta-Lactamases; D014633:Valine; D002512:Cephalothin
keywords: IMP-1 antibody; antibiotic resistance; enzyme evolution; metallo-β-lactamase; point mutation