Open AccessSubstitution of Arg‐244 by Cys or Ser in SHV‐1 and SHV‐5 β‐lactamases confers resistance to mechanism‐based inhibitors and reduces catalytic efficiency of the enzymes
Author(s) -
Giakkoupi Panagiota,
Tzelepi Eva,
Legakis Nicholas J,
Tzouvelekis Leonidas S
Publication year - 1998
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1998.tb12889.x
Subject(s) - aztreonam , mutant , enzyme , sulbactam , chemistry , biochemistry , mutagenesis , microbiology and biotechnology , biology , antibiotics , antibiotic resistance , gene , imipenem
The conserved residue Arg‐244 was substituted by the smaller uncharged amino acids Cys and Ser in SHV‐1 and SHV‐5 β‐lactamases by a PCR‐based site‐specific mutagenesis procedure. The mutant β‐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‐β‐lactams except aztreonam. The impaired catalytic activity of the mutant β‐lactamases was mainly due to the lowering of affinity for β‐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 β‐lactamase inhibitors and (b) the proper positioning of β‐lactams in the active site of SHV enzymes.