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Antimicrobial Agents and Chemotherapy, June 2002, p. 1966-1970, Vol. 46, No. 6
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.6.1966-1970.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutational Replacement of Leu-293 in the Class C Enterobacter cloacae P99 ß-Lactamase Confers Increased MIC of Cefepime

Sergei B. Vakulenko,1,3 Dasantila Golemi,2 Bruce Geryk,1 Maxim Suvorov,1 James R. Knox,4 Shahriar Mobashery,2,3,5 and Stephen A. Lerner1,3*

Departments of Medicine,1 Biochemistry and Molecular Biology,3 Institute for Drug Design, Wayne State University, Detroit, Michigan 48201,5 Department of Chemistry, Wayne State University, Detroit, Michigan 48202,2 Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 062694

Received 13 June 2001/ Returned for modification 5 October 2001/ Accepted 16 March 2002

The class C ß-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum ß-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 ß-lactamase conferring a higher MIC of cefepime (MIC, 8 µg/ml, compared with 0.5 µg/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other ß-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 ß-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (kcat/Km) of the Leu-293-Pro mutant ß-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 ß-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant ß-lactamase.

* Corresponding author. Mailing address: Division of Infectious Diseases, Harper University Hospital, 3990 John R, Detroit, MI 48201. Phone: (313) 745-9131. Fax: (313) 993-0302. E-mail: slerner{at}intmed.wayne.edu.

Antimicrobial Agents and Chemotherapy, June 2002, p. 1966-1970, Vol. 46, No. 6
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.6.1966-1970.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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