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Elucidating the role of Trp105 in the KPC-2 β-lactamase.
- Source :
-
Protein science : a publication of the Protein Society [Protein Sci] 2010 Sep; Vol. 19 (9), pp. 1714-27. - Publication Year :
- 2010
-
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.<br /> (Copyright © 2010 The Protein Society.)
- Subjects :
- Anti-Bacterial Agents chemistry
Carbapenems chemistry
Cephalosporins chemistry
Escherichia coli drug effects
Escherichia coli genetics
Gene Expression
Microbial Sensitivity Tests
Models, Molecular
Molecular Structure
Mutagenesis, Site-Directed
Penicillins chemistry
Tryptophan chemistry
Tryptophan genetics
beta-Lactamase Inhibitors
beta-Lactamases chemistry
beta-Lactamases genetics
Anti-Bacterial Agents pharmacology
Carbapenems pharmacology
Cephalosporins pharmacology
Escherichia coli enzymology
Penicillins pharmacology
Tryptophan metabolism
beta-Lactamases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1469-896X
- Volume :
- 19
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Protein science : a publication of the Protein Society
- Publication Type :
- Academic Journal
- Accession number :
- 20662006
- Full Text :
- https://doi.org/10.1002/pro.454