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 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-I 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 tazobactarn. 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 CW beta-lactamase was found to share more than 69.3% identity with the chromosamally encoded beta-lactamases of Klebsiella axytoca, Proteus vulgaris, Citrobacter diversus and the plasmid-mediated enzymes MEN-I 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. (C) 1997 Elsevier Science B.V.