Molecular evolution of a class C β-lactamase extending its substrate specificity

Abstract

Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C β-lactamase with extended substrate specificity to oxyimino β-lactam antibiotics, significantly differing from the known E. cloacae β-lactamases such as the P99 β-lactamase. The 1560 nucleotides including the GC1 β-lactamase gene were sequenced, and the amino acid sequence of the mature enzyme comprising 364 amino acids was deduced. A comparison of the amino acid sequence with those of known E. cloacae β- lactamases revealed the duplication of three amino acids at positions 208- 213, i.e. Ala-Val-Arg-Ala-Val-Arg. This duplication was attributed to a tandem duplication of a 9-nucleotide sequence. The chimeric β-lactamases produced by the chimeric genes from the GC1 and P99 β-lactamase genes indicated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion. Two mutant β-lactamases were prepared from P99 β-lactamase by site-directed mutagenesis, i.e. an Ala-Ala-Ala sequence was inserted before or after the native Ala-Val-Arg at positions 208-210. These mutant enzymes revealed that the Ala-Val-Arg located from positions 211 to 213 in the GC1 β-lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.