title: Mutational replacement of Leu-293 in the class C Enterobacter cloacae P99 beta-lactamase confers increased MIC of cefepime.
abstract: The class C beta-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum beta-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the E. cloacae P99 ampC gene, we obtained a Leu-293-Pro mutant of the P99 beta-lactamase conferring a higher MIC of cefepime (MIC, 8 microg/ml, compared with 0.5 microg/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other beta-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified E. cloacae P99 beta-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (k(cat)/K(m)) of the Leu-293-Pro mutant beta-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 beta-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant beta-lactamase.
authors.affiliation: Department of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: Sergei B
authors.initials: SB
authors.lastname: Vakulenko
authors.forename: Dasantila
authors.initials: D
authors.lastname: Golemi
authors.forename: Bruce
authors.initials: B
authors.lastname: Geryk
authors.forename: Maxim
authors.initials: M
authors.lastname: Suvorov
authors.forename: James R
authors.initials: JR
authors.lastname: Knox
authors.forename: Shahriar
authors.initials: S
authors.lastname: Mobashery
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Lerner
mesh_terms: D000077723:Cefepime; D018550:Cephalosporin Resistance; D002511:Cephalosporins / Q000494:pharmacology*; D003001:Cloning, Molecular; D016972:Enterobacter cloacae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D007930:Leucine / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D002511:Cephalosporins; D000077723:Cefepime; D001618:beta-Lactamases; D007930:Leucine