CHARACTERIZATION AND AMINO-ACID-SEQUENCE ANALYSIS OF A NEW OXYIMINO CEPHALOSPORIN-HYDROLYZING CLASS-A BETA-LACTAMASE FROM SERRATIA-FONTICOLA CUV

Serratia fonticola CUV produces two isoenzymes (forms I and II) with b eta-lactamase activity which were purified by a five-step procedure. T he isoenzymes had identical kinetic parameters and isoelectric point ( pI = 8.12), They were characterized by a specific activity towards ben zylpenicillin of 1650 U/mg, The beta-lactamase hydrolyzed benzylpenici llin, amoxycillin, ureidopenicillins, first-and second-generation ceph alosporins, Carboxypenicillins and isoxazolylpenicillins were hydrolyz ed to a lesser extent Towards cefotaxime and ceftriaxone (third-genera tion cephalosporins), the S, fonticola enzyme exhibited catalytic effi ciencies much higher than those of MEN-I and extended-spectrum TEM der ivative beta-lactamases. The beta-lactamase from S. fonticola was mark edly inhibited by beta-lactamase inhibitors such as clavulanic acid, s ulbactam and tazobactarn. The purified isoenzymes were digested by try psin, endoproteinase Asp-N and chymotrypsin. Amino acid sequence deter minations 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-termi nus, From amino acid sequence comparisons, S. fonticola CW beta-lactam ase was found to share more than 69.3% identity with the chromosamally encoded beta-lactamases of Klebsiella axytoca, Proteus vulgaris, Citr obacter diversus and the plasmid-mediated enzymes MEN-I and Toho-1. Th erefore, the oxyimino cephalosporin-hydrolyzing beta-lactamase of S. f onticola belongs to Ambler's class A. Contribution of the serine at AB L 237 in the broad-spectrum activity of these beta-lactamases is discu ssed. (C) 1997 Elsevier Science B.V.