MOLECULAR EVOLUTION OF A CLASS-C BETA-LACTAMASE EXTENDING ITS SUBSTRATE-SPECIFICITY

Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C beta-lactamase with extend ed substrate specificity to oxyimino beta-lactam antibiotics, signific antly differing from the known E. cloacae beta-lactamases such as the P99 beta-lactamase. The 1560 nucleotides including the GC1 beta-lactam ase gene were sequenced, and the amino acid sequence of the mature enz yme comprising 364 amino acids was deduced. A comparison of the amino acid sequence with those of known E. cloacae beta-lactamases revealed the duplication of three amino acids at positions 208-213, i.e. Ala-Va l-Arg-Ala-Val-Arg. This duplication was attributed to a tandem duplica tion of a 9-nucleotide sequence, The chimeric beta-lactamases produced by the chimeric genes from the GC1 and P99 beta-lactamase genes indic ated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion, Two mutant beta-lactamases were prepared from P99 beta-lactamase by site-directed mutagenesis, i.e. an Ala-Ala- Ala sequence was inserted before or after the native Ala-Val-Arg at po sitions 208-210, These mutant enzymes revealed that the Ala-Val-Arg lo cated from positions 211 to 213 in the GC1 beta-lactamase are the newl y inserted residues, and this phenomenon is independent of the charact eristics of the amino acids inserted.