title: Mutagenesis of amino acid residues in the SHV-1 beta-lactamase: the premier role of Gly238Ser in penicillin and cephalosporin resistance.
abstract: 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, Asp104Lys-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 oxyimino-cephalosporins: 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 Ser130Gly 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-1 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.
authors.affiliation: Research Service, Louis Stokes Veterans Affairs Medical Center, OH 44106, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Hujer
authors.forename: K M
authors.initials: KM
authors.lastname: Hujer
authors.forename: R A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002511:Cephalosporins / Q000494:pharmacology*; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D007711:Klebsiella pneumoniae / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010406:Penicillins / Q000494:pharmacology*; D001618:beta-Lactamases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
chemical_list: D000596:Amino Acids; D002511:Cephalosporins; D010406:Penicillins; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases