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 extended substrate specificity to oxyimino beta-lactam antibiotics, significantly differing from the known E. cloacae beta-lactamases such as the P99 beta-lactamase. The 1560 nucleotides including the GC1 beta-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 beta-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 beta-lactamases produced by the chimeric genes from the GC1 and P99 beta-lactamase genes indicated 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 positions 208-210. These mutant enzymes revealed that the Ala-Val-Arg located from positions 211 to 213 in the GC1 beta-lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.
Nukaga
M
M
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Haruta
S
S
Tanimoto
K
K
Kogure
K
K
Taniguchi
K
K
Tamaki
M
M
Sawai
T
T
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D005075:Biological Evolution; D003001:Cloning, Molecular; D016972:Enterobacter cloacae / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D047090:beta-Lactams
D000900:Anti-Bacterial Agents; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D047090:beta-Lactams; D001618:beta-Lactamases