REPLACEMENT OF SERINE-237 IN CLASS-A BETA-LACTAMASE OF PROTEUS-VULGARIS MODIFIES ITS UNIQUE SUBSTRATE-SPECIFICITY

The chromosomal beta-lactamase gene of Proteus vulgaris K1 was cloned and sequenced. The gene comprises and 813 nucleotides and codes for th e mature enzyme of 29 655 Da, comprising 271 amino acids. The K1 beta- lactamase showed 30-70% similarity, in the overall amino acid sequence , to class A beta-lactamases of Gram-negative bacteria. However, the K 1 beta-lactamase differs from most class A enzymes in having a unique substrate specificity as a cephalosporinase, its spectrum extending to even oxyiminocephalosporins. To clarify the relationship between its unique substrate specificity and specific amino acid residues, alignme nt of the amino acid sequence of the K1 beta-lactamase with those of c lass A beta-lactamases was performed, and Ala104 and Ser237 were found to be candidates. Ala104 and Ser237 were replaced with glutamic acid and alanine, respectively, which are commonly found in other class A b eta-lactamases. The substitution at position 104 had no effect on the enzyme activity or the substrate specificity. The amino acid replaceme nt at position 237, however, reduced the k(cat)/K-m value for an oxyim inocephalosporin (cefuroxime) to 17% of that in the case of the wild-t ype enzyme, whereas the mutant enzyme showed a higher k(cat)/K-m for b enzylpenicillin, 3 times, than that of the wild-type enzyme. These res ults indicated that Ser237 is one of the residues responsible for the unique substrate specificity of the P. vulgaris beta-lactamase.