AN ENGINEERED STAPHYLOCOCCUS-AUREUS PC1 BETA-LACTAMASE THAT HYDROLYZES 3RD-GENERATION CEPHALOSPORINS

The beta-lactamase from Staphylococcus aureus PC1 has been cloned into an Escherichia coli vector for site-directed mutagenesis and high-lev el protein expression. A mutant enzyme has been produced in which Ala2 38 is replaced by a serine, and Ile239 is deleted (A238S:I239del). The engineered enzyme hydrolyses third-generation cephalosporins substant ially more rapidly than the parental enzyme does, while hydrolysis of benzylpenicillin is slower with the mutant than with the wild-type and native enzymes. The mutant beta-lactamase has been crystallized and t he structure determined and refined at 2.8 Angstrom resolution. The di sposition of the beta-strand which forms the side of the active site i s altered in comparison with the native S. aureus beta-lactamase struc ture, widening the active site cleft and providing space to accommodat e the bulky side-chains of the third-generation cephalosporins.