来自蜡样芽孢杆菌的β-内酰胺酶I。结构与定点诱变
beta-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis.
作者信息
Madgwick P J, Waley S G
机构信息
Sir William Dunn School of Pathology, University of Oxford, U.K.
出版信息
Biochem J. 1987 Dec 15;248(3):657-62. doi: 10.1042/bj2480657.
Abstract
The sequence of the gene for beta-lactamase I from Bacillus cereus 569/H has been redetermined. Oligonucleotide-directed mutagenesis has been carried out, and the effects of the changes on the ampicillin-resistance of Escherichia coli TG1 expressing the mutant genes have been studied. Lysine-73, close to the active-site serine-70 and a highly-conserved residue, has been converted into arginine. This change had a large effect on activity, but did not abolish it. An even larger effect was found in the mutant in which glutamate-166 had been converted into glutamine; this had little or no activity. On the other hand, the conversion of glutamate-168 into aspartate gave fully active enzyme. Glutamate-166 is an invariant residue, but glutamate-168 is not. Alanine-123 has been replaced by cysteine, to give active enzyme; this change forms part of the plan to introduce a disulphide bond into the enzyme.
摘要
蜡样芽孢杆菌569/H的β-内酰胺酶I基因序列已重新测定。已进行了寡核苷酸定向诱变，并研究了这些变化对表达突变基因的大肠杆菌TG1氨苄青霉素抗性的影响。靠近活性位点丝氨酸-70且高度保守的赖氨酸-73已被转化为精氨酸。这一变化对活性有很大影响，但并未使其丧失。在谷氨酸-166已被转化为谷氨酰胺的突变体中发现了更大的影响；该突变体几乎没有或没有活性。另一方面，谷氨酸-168转化为天冬氨酸得到了完全有活性的酶。谷氨酸-166是一个不变残基，但谷氨酸-168不是。丙氨酸-123已被半胱氨酸取代，以得到有活性的酶；这一变化是将二硫键引入该酶计划的一部分。
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