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

M Nukaga·S Haruta·K Tanimoto·K Kogure·K Taniguchi·M Tamaki·T Sawai
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Abstract

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.

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Amino Acid SequenceAnti-Bacterial AgentsBase SequenceBinding SitesBiological EvolutionCloning, MolecularEnterobacter cloacaeEscherichia coliHumansMicrobial Sensitivity TestsMolecular Sequence DataMutagenesis, Site-DirectedProtein ConformationRecombinant Fusion ProteinsRecombinant ProteinsSequence Homology, Amino AcidSubstrate Specificitybeta-Lactamasesbeta-LactamsComparative StudyJournal ArticleResearch Support, Non-U.S. Gov't

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M Nukaga, S Haruta, K Tanimoto, K Kogure, K Taniguchi, M Tamaki, T Sawai. 1995-03-17. Molecular evolution of a class C beta-lactamase extending its substrate specificity.. https://doi.org/10.1074/jbc.270.11.5729

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