Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 β-lactamases
Open Access
- 1 January 1999
- journal article
- research article
- Published by Oxford University Press (OUP) in Journal of Antimicrobial Chemotherapy
- Vol. 43 (1), 23-29
- https://doi.org/10.1093/jac/43.1.23
Abstract
In SHV-type β-lactamases, position 276 (in Ambler's numbering scheme) is occupied by an asparagine (Asn) residue. The effect on SHV-1 β-lactamase and its extended-spectrum derivative SHV-5 of substituting an aspartic acid (Asp) residue for Asn276 was studied. Mutations were introduced by a PCR-based site-directed mutagenesis procedure. Wild-type SHV-1 and -5 β-lactamases and their respective Asn276→Asp mutants were expressed under isogenic conditions by cloning the respective bla genes into the pBCSK(+) plasmid and transforming Escherichia coliDH5α. Determination of IC50 showed that SHV-1(Asn276→Asp), compared with SHV-1, was inhibited by 8- and 8.8-fold higher concentrations of clavulanate and tazobactam respectively. Replacement of Asn276 by Asp in SHV-5 β-lactamase caused a ten-fold increase in the IC50 of clavulanate; the increases in the IC50s of tazobactam and sulbactam were 10- and 5.5-fold, respectively. β-Lactam susceptibility testing showed that both Asn276→Asp mutant enzymes, compared with the parental β-lactamases, conferred slightly lower levels of resistance to penicillins (amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of the expanded-spectrum oxyimino β-lactams tested (cefotaxime, ceftriaxone and aztreonam). The MICs of ceftazidime remained unaltered, while those of cefepime and cefpirome were slightly elevated in the clones producing the mutant β-lactamases. The latter clones were also less susceptible to penicillin-inhibitor combinations. Asn276→Asp mutation was associated with changes in the substrate profiles of SHV-1 and SHV-5 enzymes. Based on the structure of TEM-1 β-lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5 are discussed.Keywords
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