title: N152G, -S, and -T substitutions in CMY-2 β-lactamase increase catalytic efficiency for cefoxitin and inactivation rates for tazobactam.
abstract: Class C cephalosporinases are a growing threat, and clinical inhibitors of these enzymes are currently unavailable. Previous studies have explored the role of Asn152 in the Escherichia coli AmpC and P99 enzymes and have suggested that interactions between C-6' or C-7' substituents on penicillins or cephalosporins and Asn152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of Asn152 in the clinically important CMY-2 cephalosporinase with substrates and inhibitors. Mutagenesis of CMY-2 at position 152 yields functional mutants (N152G, -S, and -T) that exhibit improved penicillinase activity and retain cephamycinase activity. We also tested whether the position 152 substitutions would affect the inactivation kinetics of tazobactam, a class A β-lactamase inhibitor with in vitro activity against CMY-2. Using standard assays, we showed that the N152G, -S, and -T variants possessed increased catalytic activity against cefoxitin compared to the wild type. The 50% inhibitory concentration (IC50) for tazobactam improved dramatically, with an 18-fold reduction for the N152S mutant due to higher rates of enzyme inactivation. Modeling studies have shown active-site expansion due to interactions between Y150 and S152 in the apoenzyme and the Michaelis-Menten complex with tazobactam. Substitutions at N152 might become clinically important as new class C β-lactamase inhibitors are developed.
authors: Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. marion.skalweit@case.edu
authors: Marion J
authors: MJ
authors: Skalweit
authors: Mei
authors: M
authors: Li
authors: Benjamin C
authors: BC
authors: Conklin
authors: Magdalena A
authors: MA
authors: Taracila
authors: Rebecca A
authors: RA
authors: Hutton
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D002384:Catalysis; D002440:Cefoxitin / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000078142:Tazobactam; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D002440:Cefoxitin; D010397:Penicillanic Acid; C103022:beta-lactamase CMY-2; D001618:beta-Lactamases; D000078142:Tazobactam