title: Elucidating the role of Trp105 in the KPC-2 β-lactamase.
abstract: The molecular basis of resistance to β-lactams and β-lactam-β-lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective β-lactam therapy. Recent crystal structures of KPC-2 and other class A β-lactamases suggest that Ambler position Trp105 may be of importance in binding β-lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC-2 by conducting site-saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, -Tyr, -Asn, and -His KPC-2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC-2. Interestingly, most of the variants showed increased MICs to ampicillin-clavulanic acid but not to ampicillin-sulbactam or piperacillin-tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, -Asn, -Leu, and -Val) were studied in detail. Consistent with the MIC data, the Trp105Phe β-lactamase displayed improved catalytic efficiencies, k(cat)/K(m), toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k(cat)/K(m) toward cefotaxime and imipenem when compared to WT β-lactamase. The Trp105Asn variant exhibited increased K(m)s for all substrates. In contrast, the Trp105Leu and -Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k(cat)/K(m)) for all substrates. With respect to clavulanic acid, the K(i)s and partition ratios were increased for the Trp105Phe, -Asn, and -Val variants. We conclude that interactions between Trp105 of KPC-2 and the β-lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in β-lactam and β-lactamase inhibitor discrimination.
authors.affiliation: Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Papp-Wallace
authors.forename: Magdalena
authors.initials: M
authors.lastname: Taracila
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Wallace
authors.forename: Kristine M
authors.initials: KM
authors.lastname: Hujer
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.forename: John M
authors.initials: JM
authors.lastname: Hornick
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D015780:Carbapenems / Q000737:chemistry / Q000494:pharmacology*; D002511:Cephalosporins / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010406:Penicillins / Q000737:chemistry / Q000494:pharmacology*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D002511:Cephalosporins; D010406:Penicillins; D065093:beta-Lactamase Inhibitors; D014364:Tryptophan; C491478:beta-lactamase KPC-2; D001618:beta-Lactamases