β-lactamase I from Bacillus cereus: structure and site-directed mutagenesis
β-lactamase I from Bacillus cereus: structure and site-directed mutagenesis
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来自蜡样芽孢杆菌的 β-内酰胺酶 I：结构和定点诱变
DOI：
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发表时间：
1987
期刊：
影响因子：
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通讯作者：
S. G. Waley
中科院分区：
文献类型：
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作者：
P. Madgwick;S. G. Waley
蜡状芽孢杆菌 569/H 的 β-内酰胺酶 I 基因序列已被重新确定。已经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的氨苄青霉素抗性的影响。赖氨酸73接近活性位点丝氨酸70，是高度保守的残基，已被转化为精氨酸。这一变化对活动产生了很大影响，但并没有废除它。在 166 号谷氨酸转化为谷氨酰胺的突变体中发现了更大的影响。这几乎没有活动。另一方面，谷氨酸168转化为天冬氨酸使酶具有完全活性。 Glutamate-166 是不变残基，但 glutamate-168 不是。 123丙氨酸被半胱氨酸取代，产生活性酶；这一变化是在酶中引入二硫键的计划的一部分。
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 Alanine-123 has been replaced by cysteine, to give active enzyme; this change forms part of the plan to introduce a disulphide bond into the enzyme.