Molecular Evolution of a Class C β-Lactamase Extending Its Substrate Specificity (∗)

• Published in: The Journal of biological chemistry Vol. 270; no. 11; pp. 5729 - 5735
• Main Authors: Nukaga, Michiyoshi, Haruta, Shin, Tanimoto, Kyoko, Kogure, Keiko, Taniguchi, Kazuo, Tamaki, Mami, Sawai, Tetsuo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17-03-1995
• Subjects: Amino Acid Sequence; Anti-Bacterial Agents - pharmacology; Base Sequence; beta-Lactamases - chemistry; beta-Lactamases - genetics; beta-Lactamases - metabolism; beta-Lactams; Binding Sites; Biological Evolution; Cloning, Molecular; Enterobacter cloacae; Enterobacter cloacae - enzymology; Enterobacter cloacae - genetics; Enterobacter cloacae - isolation & purification; Escherichia coli - drug effects; Escherichia coli - enzymology; Escherichia coli - genetics; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.11.5729

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.