title: Characterization and amino acid sequence analysis of a new oxyimino cephalosporin-hydrolyzing class A beta-lactamase from Serratia fonticola CUV.
abstract: Serratia fonticola CUV produces two isoenzymes (forms I and II) with beta-lactamase activity which were purified by a five-step procedure. The isoenzymes had identical kinetic parameters and isoelectric point (pI = 8.12). They were characterized by a specific activity towards benzylpenicillin of 1650 U/mg. The beta-lactamase hydrolyzed benzylpenicillin, amoxycillin, ureidopenicillins, first- and second-generation cephalosporins. Carboxypenicillins and isoxazolylpenicillins were hydrolyzed to a lesser extent. Towards cefotaxime and ceftriaxone (third-generation cephalosporins), the S. fonticola enzyme exhibited catalytic efficiencies much higher than those of MEN-1 and extended-spectrum TEM derivative beta-lactamases. The beta-lactamase from S. fonticola was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. The purified isoenzymes were digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino acid sequence determinations of the resulting peptides allowed the alignment of 267 amino acid residues (Swiss-Prot, accession number P 80545) for form I beta-lactamase. Form II is five residues shorter than form I at its N-terminus. From amino acid sequence comparisons, S. fonticola CUV beta-lactamase was found to share more than 69.3% identity with the chromosomally encoded beta-lactamases of Klebsiella oxytoca, Proteus vulgaris, Citrobacter diversus and the plasmid-mediated enzymes MEN-1 and Toho-1. Therefore, the oxyimino cephalosporin-hydrolyzing beta-lactamase of S. fonticola belongs to Ambler's class A. Contribution of the serine at ABL 237 in the broad-spectrum activity of these beta-lactamases is discussed.
authors.affiliation: Muséum National Histoire Naturelle, CNRS URA 401, IFR 63, Paris, France. Peduzzi@infobiogen.fr
authors.forename: J
authors.initials: J
authors.lastname: Péduzzi
authors.forename: S
authors.initials: S
authors.lastname: Farzaneh
authors.forename: A
authors.initials: A
authors.lastname: Reynaud
authors.forename: M
authors.initials: M
authors.lastname: Barthélémy
authors.forename: R
authors.initials: R
authors.lastname: Labia
mesh_terms: D000595:Amino Acid Sequence; D002511:Cephalosporins / Q000378:metabolism*; D002918:Chymotrypsin; D004352:Drug Resistance, Microbial; D010450:Endopeptidases; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012705:Serratia / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D014357:Trypsin; D001618:beta-Lactamases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002511:Cephalosporins; D007527:Isoenzymes; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D001618:beta-Lactamases