title: Elucidating the Role of Residue 67 in IMP-Type Metallo-β-Lactamase Evolution.
abstract: Antibiotic resistance in bacteria is ever changing and adapting, as once-novel β-lactam antibiotics are losing their efficacy, primarily due to the production of β-lactamases. Metallo-β-lactamases (MBLs) efficiently inactivate a broad range of β-lactam antibiotics, including carbapenems, and are often coexpressed with other antibacterial resistance factors. The rapid dissemination of MBLs and lack of novel antibacterials pose an imminent threat to global health. In an effort to better counter these resistance-conferring β-lactamases, an investigation of their natural evolution and resulting substrate specificity was employed. In this study, we elucidated the effects of different amino acid substitutions at position 67 in IMP-type MBLs on the ability to hydrolyze and confer resistance to a range of β-lactam antibiotics. Wild-type β-lactamases IMP-1 and IMP-10 and mutants IMP-1-V67A and IMP-1-V67I were characterized biophysically and biochemically, and MICs for Escherichia coli cells expressing these enzymes were determined. We found that all variants exhibited catalytic efficiencies (kcat/Km) equal to or higher than that of IMP-1 against all tested β-lactams except penicillins, against which IMP-1 and IMP-1-V67I showed the highest kcat/Km values. The substrate-specific effects of the different amino acid substitutions at position 67 are discussed in light of their side chain structures and possible interactions with the substrates. Docking calculations were employed to investigate interactions between different side chains and an inhibitor used as a β-lactam surrogate. The differences in binding affinities determined experimentally and computationally seem to be governed by hydrophobic interactions between residue 67 and the inhibitor and, by inference, the β-lactam substrates.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, California, USA.
authors.forename: Alecander E
authors.initials: AE
authors.lastname: LaCuran
authors.affiliation: Department of Biological Sciences, California State Polytechnic University, Pomona, California, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Pegg
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, California, USA.
authors.forename: Eleanor M
authors.initials: EM
authors.lastname: Liu
authors.affiliation: Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.affiliation: Pharmaceutical Informatics Institute, School of Pharmaceutical Sciences, Zhejiang University, Zhejiang, People's Republic of China.
authors.forename: Ni
authors.initials: N
authors.lastname: Ai
authors.affiliation: Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, and Division of Chem Informatics, Biomedical Informatics Shared Resource, Rutgers-Cancer Institute of New Jersey, Piscataway, New Jersey, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Welsh
authors.affiliation: Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, USA Departments of Medicine, Pharmacology, Biochemistry, and Molecular Biology & Microbiology, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, California, USA poelschlaeger@westernu.edu.
authors.forename: Peter
authors.initials: P
authors.lastname: Oelschlaeger
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D019143:Evolution, Molecular; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D062105:Molecular Docking Simulation; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014633:Valine / Q000737:chemistry* / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047090:beta-Lactams / Q000737:chemistry* / Q000145:classification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D047090:beta-Lactams; D010649:Phenylalanine; C409201:beta-lactamase IMP-1; D001618:beta-Lactamases; D014633:Valine