Elucidating the role of Trp105 in the KPC-2 beta-lactamase

作者：Papp Wallace Krisztina M; Taracila Magdalena; Wallace Christopher J; Hujer Kristine M; Bethel Christopher R; Hornick John M; Bonomo Robert A^*

来源：Protein Science, 2010, 19(9): 1714-1727.

DOI：10.1002/pro.454

• beta-lactamase
• KPC-2
• carbapenemase
• carbapenem discrimination
• clavulanic acid

The molecular basis of resistance to beta-lactams and beta-lactam-beta-lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective beta-lactam therapy. Recent crystal structures of KPC-2 and other class A beta-lactamases suggest that Ambler position Trp105 may be of importance in binding beta-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 beta-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 beta-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 beta-lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in beta-lactam and beta-lactamase inhibitor discrimination.

• 出版日期2010-9