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J. Biol. Chem., Vol. 278, Issue 52, 52724-52729, December 26, 2003

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Understanding Resistance to -Lactams and -Lactamase Inhibitors in the SHV -Lactamase

LESSONS FROM THE MUTAGENESIS OF SER-130^*

Marion S. Helfand, Christopher R. Bethel, Andrea M. Hujer, Kristine M. Hujer, Vernon E. Anderson¶, and Robert A. Bonomo||

From the Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, and ^¶Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Bacterial resistance to -lactam/-lactamase inhibitor combinations by single amino acid mutations in class A -lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A -lactamases, alterations at Ser-130 confer resistance to inactivation by the -lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 -lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G -lactamase expressed in Escherichia coli. Kinetic analysis of the S130G -lactamase demonstrated a significant elevation in apparent K_m and a reduction in k_cat/K_m for ampicillin. Marked increases in the dissociation constant for the preacylation complex, K_I, of clavulanic acid (SHV-1, 0.14 µM; S130G, 46.5 µM) and tazobactam (SHV-1, 0.07 µM; S130G, 4.2 µM) were observed. In contrast, the k_inacts of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for -lactams and -lactamase inhibitors. These results underscore the mechanistic versatility of class A -lactamases and have implications for the design of novel -lactamase inhibitors.

Received for publication, June 9, 2003 , and in revised form, October 8, 2003.

^* This work was supported by grants from Department of Veterans Affairs Career Development Award (to M. S. H.) and the Veterans Affairs Medical Center Merit Review Program (to R. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplementary Fig. 1.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed: Infectious Disease Section, Louis Stokes Cleveland Dept. of Veterans Affairs Medical Ctr., 10701 East Blvd., Cleveland, OH 44106. Tel.: 216-791-3800 (ext. 4399); Fax: 216-231-3482; E-mail: robert.bonomo{at}med.va.gov.

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