Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 beta-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic efficiency of the enzymes.
The conserved residue Arg-244 was substituted by the smaller uncharged amino acids Cys and Ser in SHV-1 and SHV-5 beta-lactamases by a PCR-based site-specific mutagenesis procedure. The mutant beta-lactamases displayed decreased susceptibility to clavulanate and, to a lesser extent, to tazobactam and sulbactam. As shown in comparative MIC determinations, R244C and R244S enzymes retained a residual penicillinase activity while their activity towards cephalosporins was drastically diminished. The respective SHV-5 mutants were unable to hydrolyze oxyimino-beta-lactams except aztreonam. The impaired catalytic activity of the mutant beta-lactamases was mainly due to the lowering of affinity for beta-lactam substrates. The above alterations were more pronounced in the R244C mutants. These results provide information on the mode of involvement of Arg-244 in (a) inactivation by beta-lactamase inhibitors and (b) the proper positioning of beta-lactams in the active site of SHV enzymes.
Giakkoupi
P
P
Department of Bacteriology, Hellenic Pasteur Institute, Athens, Greece.
Tzelepi
E
E
Legakis
N J
NJ
Tzouvelekis
L S
LS
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents; D001120:Arginine; D019818:Clavulanic Acid; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D010403:Penicillin Resistance; D012694:Serine; D001618:beta-Lactamases / Q000235:genetics*
D000900:Anti-Bacterial Agents; D019818:Clavulanic Acid; D012694:Serine; D001120:Arginine; C054882:beta-lactamase PIT-2; C117193:beta-lactamase SHV-5; D001618:beta-Lactamases; D003545:Cysteine