Mutagenesis of amino acid residues in the SHV-1 β-lactamase:: the premier role of Gly238Ser in penicillin and cephalosporin resistance

The recent availability of the SHV-1 beta -lactamase crystal structure provides a framework for the understanding of the functional role of amino acid residues in this enzyme. To that end, we have constructed by site-directed mutagenesis 18 variants of the SHV beta -lactamase: an extended spectrum group: Gly238Ser, Gly238Ser-Glu240Lys, Asp 104Lys-Gly238Ser, Asp104Lys-Thr235Ser-Gly238Ser. Asp179Asn, Arg164His, and Arg164Ser: an inhibitor resistant group: Arg244Ser. Met69Ile, Met69Leu, and Ser130Gly; mutants that are synergistic with those that confer resistance to oxyiminocephalosporins: Asp104Glu, Asp104Lys, Glu240Lys, and Glu240Gln; and structurally conserved mutants. Thr235Ser, Thr235Ala and Glu166Ala. Among the extended spectrum group the combination of high-level ampicillin and cephalosporin resistance was demonstrated in the Escherichia coli DH10B strains possessing the Gly238Ser mutation: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, and Asp104Lys-Thr235Ser-Gly238Ser. Of the inhibitor resistant group, the Ser 130Gly mutant was the most resistant to ampicillin/clavulanate. Using a polyclonal anti-SHV antibody, we assayed steady state protein expression levels of the SHV beta -lactamase variants. Mutants with the Gly238Ser substitution were among the most highly expressed. The Gly238Ser substitution resulted in an improved relative k(cat)/K-m value for cephaloridine and oxyimino-cephalosporins compared to SHV-I and Met69Ile. In our comparative survey, the Gly238Ser and extended spectrum beta -lactamase variants containing this substitution exhibited the greatest substrate versatility against penicillins and cephalosporins and greatest protein expression. This defines a unique role of Gly238Ser in broad-spectrum beta -lactam resistance in this family of class A beta -lactamases. (C) 2001 Elsevier Science B.V. All rights reserved.