| Abstract: | The duplicative mutation of an Ala-Val-Arg sequence at positions 208 to 210 in the loop structure of Enterobacter cloacae class C β-lactamase caused substrate specificity extension to oxyimino β-lactam antibiotics and this chromosomal mutation provided bacterial cells with high resistance to the β-lactams (M. Nukaga et al, 1995, J. Biol. Chem. 270, 5729-5735). In order to confirm the universality of this phenomenon among other class C β-lactamases, the duplicative mutation was applied to a class C β-lactamase of Citrobacter freundii, which has 74% homology to the E. cloacae β-lactamase amino acid sequence. The counterpart sequence to the Ala-Val-Arg of the E. cloacae enzyme in C. freundii β-lactamase was identified to be Pro-Val-His. A Pro-Val-His sequence was inserted just after the native Pro-Val-His sequence at positions 208 to 210 in the C. freundii β-lactamase. The resulting mutant of C. freundii β-lactamase obtained a striking characteristic that we expected, showing substrate specificity extension to oxyimino β-lactams. Nearly the same result was obtained with the insertion of an Ala-Val-Arg sequence after the native Pro-Val-His sequence. These results indicate that structural modification of this locus commonly induces modification of the substrate specificity to unfavorable substrates for many chromosomal class C β-lactamases produced by Gram-negative bacteria. |
