SEQUENCE-ANALYSIS AND ENZYME-KINETICS OF THE L2 SERINE BETA-LACTAMASEFROM STENOTROPHOMONAS-MALTOPHILIA

The L2 serine active-site beta-lactamase from Stenotrophomonas maltoph ilia has been classified as a clavulanic acid-sensitive cephalosporina se. The gene encoding this enzyme from S. maltophilia 1275 IID has bee n cloned on a 3.3-kb fragment into pK18 under the control of a Ptac pr omoter to generate recombinant plasmid pUB5840; when expressed in Esch erichia coli, this gene confers resistance to cephalosporins and penic illins, Sequence analysis has revealed an open reading frame (ORF) of 909 bp with a GC content of 71.6%, comparable to that of the L1 metall o-beta-lactamase gene (68.4%) from the same bacterium, The ORF encodes an unmodified protein of 303 amino acids with a predicted molecular m ass of 31.5 kDa, accommodating a putative leader peptide of 27 amino a cids, Comparison of the amino acid sequence with those of other P-lact amases showed it to be most closely related (54% identity) to the BLA- A beta-lactamase from Yersinia enterocolitica. Sequence identity is mo st obvious near the STXK active-site motif and the SDN loop motif comm on to all serine active-site penicillinases. Sequences outside the con served regions display low homology with comparable regions of other c lass A penicillinases. Kinetics of the enzyme from the cloned gene dem onstrated an increase in activity with cefotaxime but markedly less ac tivity with imipenem than previously reported, Hence, the S. maltophil ia L2 beta-lactamase is an inducible Ambler class A beta-lactamase whi ch would account for the sensitivity to clavulanic acid.