Kocabiyik S, Mullins C, Breeding C, Perlin M H
Dept. of Biology, University of Louisville, KY 40292.
SAAS Bull Biochem Biotechnol. 1992 Jan;5:58-63.
Mutant strains containing APH(3')-II were constructed via site-directed mutagenesis of the cloned gene and by random mutagenesis of a strain containing the APH(3')-II gene on a conjugative plasmid. Substitutions at highly conserved amino acid residues produced APH(3') enzymes which in general showed reduced activity and conferred reduced levels of resistance to their substrates. Substitutions at Tyr 218 altered substrate specificity for the enzymes. Random mutagenesis produced plasmid-borne mutations conferring amikacin resistance. Two of these mutations appeared to be localized to the APH(3')-II structural gene.
通过对克隆基因进行定点诱变以及对携带接合质粒上 APH(3')-II 基因的菌株进行随机诱变，构建了含有 APH(3')-II 的突变菌株。高度保守氨基酸残基处的取代产生了 APH(3') 酶，这些酶总体上活性降低，对其底物的抗性水平也降低。Tyr 218 处的取代改变了酶的底物特异性。随机诱变产生了赋予阿米卡星抗性的质粒携带突变。其中两个突变似乎定位于 APH(3')-II 结构基因。
