工程改造嗜松假丝酵母木糖还原酶以提高NADH的利用率：多个侧链替换的拮抗作用及定点突变体在模拟体内条件下的性能
Engineering Candida tenuis Xylose reductase for improved utilization of NADH: antagonistic effects of multiple side chain replacements and performance of site-directed mutants under simulated in vivo conditions.
作者信息
Petschacher Barbara, Nidetzky Bernd
机构信息
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
出版信息
Appl Environ Microbiol. 2005 Oct;71(10):6390-3. doi: 10.1128/AEM.71.10.6390-6393.2005.
Abstract
Six single- and multiple-site variants of Candida tenuis xylose reductase that were engineered to have side chain replacements in the coenzyme 2'-phosphate binding pocket were tested for NADPH versus NADH selectivity (R(sel)) in the presence of physiological reactant concentrations. The experimental R(sel) values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274-->Arg and Arg-280-->His substitutions, which individually improved wild-type R(sel) 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.
摘要
对光滑假丝酵母木糖还原酶的六个单点和多点变体进行了测试，这些变体经设计在辅酶2'-磷酸结合口袋中具有侧链置换，在生理反应物浓度存在的情况下检测其对NADPH与NADH的选择性（R(sel)）。实验得到的R(sel)值与描述混合交替辅酶利用的动力学机制的预测结果非常吻合。赖氨酸-274→精氨酸和精氨酸-280→组氨酸替换分别使野生型R(sel)提高了50倍和20倍，当它们组合在一个双突变体中时具有相反的结构效应。
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