Amino acid sequence requirements at residues 69 and 238 for the SME-1 beta-lactamase to confer resistance to beta-lactam antibiotics.
Carbapenem antibiotics have been used to counteract resistant strains of bacteria harboring beta-lactamases and extended-spectrum beta-lactamases. Four enzymes from the class A group of beta-lactamases, NMC-A, IMI-1, SME-1, and KPC-1, efficiently hydrolyze carbapenem antibiotics. Sequence comparisons and structural information indicate that cysteines at amino acid residues 69 and 238, which are conserved in all four of these enzymes, form a disulfide bond that is unique to these beta-lactamases. To test whether this disulfide bond is required for catalytic activity, the codons for residues Cys69 and Cys238 were randomized individually and simultaneously by PCR-based mutagenesis to create random replacement libraries for these positions. Mutants that were able to confer resistance to ampicillin, imipenem, or cefotaxime were selected from these libraries. The results indicate that positions Cys69 and Cys238 are critical for hydrolysis of all of the antibiotics tested, suggesting that the disulfide bond is generally required for this enzyme to catalyze the hydrolysis of beta-lactam antibiotics.
Majiduddin
Fahd K
FK
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Palzkill
Timothy
T
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015780:Carbapenems / Q000494:pharmacology; D002511:Cephalosporins / Q000737:chemistry; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D015723:Gene Library; D006868:Hydrolysis; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010406:Penicillins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D015780:Carbapenems; D002511:Cephalosporins; D003062:Codon; D004220:Disulfides; D010406:Penicillins; C094219:beta-lactamase Sme-1; D001618:beta-Lactamases