title: Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 beta-lactamases.
abstract: In SHV-type beta-actamases, position 276 (in Ambler's numbering scheme) is occupied by an asparagine (Asn) residue. The effect on SHV-1 beta-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 beta-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 coli DH5alpha. 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 beta-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. Beta-lactam susceptibility testing showed that both Asn276-->Asp mutant enzymes, compared with the parental beta-lactamases, conferred slightly lower levels of resistance to penicillins (amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of the expanded-spectrum oxyimino beta-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 beta-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 beta-lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5 are discussed.
authors.affiliation: Department of Bacteriology, Hellenic Pasteur Institute, Athens, Greece.
authors.forename: P
authors.initials: P
authors.lastname: Giakkoupi
authors.forename: E
authors.initials: E
authors.lastname: Tzelepi
authors.forename: N J
authors.initials: NJ
authors.lastname: Legakis
authors.forename: L S
authors.initials: LS
authors.lastname: Tzouvelekis
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001216:Asparagine; D001224:Aspartic Acid; D019818:Clavulanic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D013407:Sulbactam / Q000494:pharmacology; D000078142:Tazobactam; D018440:beta-Lactam Resistance / Q000235:genetics; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D019818:Clavulanic Acid; D001224:Aspartic Acid; D001216:Asparagine; D010397:Penicillanic Acid; C054882:beta-lactamase PIT-2; C117193:beta-lactamase SHV-5; D001618:beta-Lactamases; D013407:Sulbactam; D000078142:Tazobactam