Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C -lactamase with extended substrate specificity to oxyimino -lactam antibiotics, significantly differing from the known E. cloacae -lactamases such as the P99 -lactamase. The 1560 nucleotides including the GC1 -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 -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 -lactamases produced by the chimeric genes from the GC1 and P99 -lactamase genes indicated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion. Two mutant -lactamases were prepared from P99 -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 -lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.

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