Dataset Information

Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-?-lactamase active site.

ABSTRACT: Metallo-?-lactamases catalyze the hydrolysis of a broad range of ?-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ?-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ?-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ?-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 ?M. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.

SUBMITTER: Horton LB

PROVIDER: S-EPMC3486559 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-β-lactamase active site.

Horton Lori B LB Shanker Sreejesh S Mikulski Rose R Brown Nicholas G NG Phillips Kevin J KJ Lykissa Ernest E Venkataram Prasad B V BV Palzkill Timothy T

Antimicrobial agents and chemotherapy 20120820 11

Metallo-β-lactamases catalyze the hydrolysis of a broad range of β-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 β-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of β-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme ...[more]

PMID: 22908171

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Project description:We recently detected a novel variant of an IMP-type metallo-β-lactamase gene (blaIMP-68) from meropenem-resistant but imipenem-susceptible Klebsiella pneumoniae TA6363 isolated in Tokyo, Japan. blaIMP-68 encodes a Ser262Gly point mutant of IMP-11, and transformation experiments showed that blaIMP-68 increased the MIC of carbapenems in recipient strains, whereas the MIC of imipenem was not greatly increased relative to that of other carbapenems, including meropenem. Kinetics experiments showed that IMP-68 imipenem-hydrolyzing activity was lower than that for other carbapenems, suggesting that the antimicrobial susceptibility profile of TA6363 originated from IMP-68 substrate specificity. Whole-genome sequencing showed that blaIMP-68 is harbored by the class 1 integron located on the IncL/M plasmid pTMTA63632 (88,953 bp), which was transferable via conjugation. The presence of plasmid-borne blaIMP-68 is notable, because it conferred antimicrobial resistance to carbapenems, except for imipenem, on Enterobacteriaceae and will likely affect treatment plans using antibacterial agents in clinical settings.IMPORTANCE IMP-type metallo-β-lactamases comprise one group of the "Big 5" carbapenemases. Here, a novel blaIMP-68 gene encoding IMP-68 (harboring a Ser262Gly point mutant of IMP-11) was discovered from meropenem-resistant but imipenem-susceptible Klebsiella pneumoniae TA6363. The Ser262Gly substitution was previously identified as important for substrate specificity according to a study of other IMP variants, including IMP-6. We confirmed that IMP-68 exhibited weaker imipenem-hydrolyzing activity than that for other carbapenems, demonstrating that the antimicrobial susceptibility profile of TA6363 originated from IMP-68 substrate specificity, with this likely to affect treatment strategies using antibacterial agents in clinical settings. Notably, the carbapenem resistance conferred by IMP-68 was undetectable based on the MIC of imipenem as a carbapenem representative, which demonstrates a comparable antimicrobial susceptibility profile to IMP-6-producing Enterobacteriaceae that previously spread in Japan due to lack of awareness of its existence.

Dataset Information

Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-?-lactamase active site.

Publications

Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-β-lactamase active site.

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