Zawadzke L E, Smith T J, Herzberg O
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
Protein Eng. 1995 Dec;8(12):1275-85. doi: 10.1093/protein/8.12.1275.
The beta-lactamase from Staphylococcus aureus PC1 has been cloned into an Escherichia coli vector for site-directed mutagenesis and high-level protein expression. A mutant enzyme has been produced in which Ala238 is replaced by a serine, and Ile239 is deleted (A238S:I239del). The engineered enzyme hydrolyses third-generation cephalosporins substantially more rapidly than the parental enzyme does, while hydrolysis of benzylpenicillin is slower with the mutant than with the wild-type and native enzymes. The mutant beta-lactamase has been crystallized and the structure determined and refined at 2.8 A resolution. The disposition of the beta-strand which forms the side of the active site is altered in comparison with the native S. aureus beta-lactamase structure, widening the active site cleft and providing space to accommodate the bulky side-chains of the third-generation cephalosporins.
金黄色葡萄球菌PC1的β-内酰胺酶已被克隆到大肠杆菌载体中，用于定点诱变和高水平蛋白质表达。已产生一种突变酶，其中丙氨酸238被丝氨酸取代，异亮氨酸239缺失（A238S:I239del）。与亲本酶相比，工程酶水解第三代头孢菌素的速度要快得多，而突变体对苄青霉素的水解速度比野生型和天然酶慢。突变β-内酰胺酶已结晶，并在2.8埃分辨率下确定和完善了其结构。与天然金黄色葡萄球菌β-内酰胺酶结构相比，形成活性位点一侧的β-链的排列发生了改变，扩大了活性位点裂隙，并为容纳第三代头孢菌素的庞大侧链提供了空间。
