Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes
Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes
复制标题
DOI：
10.1093/protein/gzv057
复制
发表时间：
2016-01-01
影响因子：
2.4
通讯作者：
Arnold, F. H.
中科院分区：
文献类型：
--
作者：
Cahn, J. K. B.;Baumschlager, A.;Arnold, F. H.
NAD（P）H依赖性酶在代谢和细胞过程中普遍存在，并且在制药和工业应用中也具有极大的兴趣。在这里，我们提出了一种结构导向的酶工程策略，用于改善NAD（P）H依赖性酶对天然或天然样反应的催化性能，使用突变的酶的腺嘌呤结合口袋，远端的催化位点。筛选单位点饱和诱变文库鉴定了在10种酶中的7种中使催化效率增加高达10倍的突变。本研究中改进的酶代表三种不同的辅因子结合折叠（Rossmann、DHQS样和FAD/NAD结合），并利用NADH和NADPH。结构和生物化学分析表明，改善的活动伴随着最小的变化，在其他属性（协同性，热稳定性，pH最佳，解偶联），和两个酶（ScADH 6和EcFucO）的初步测试显示在大肠杆菌中的功能改善。
NAD(P)H-dependent enzymes are ubiquitous in metabolism and cellular processes and are also of great interest for pharmaceutical and industrial applications. Here, we present a structure-guided enzyme engineering strategy for improving catalytic properties of NAD(P)H-dependent enzymes toward native or native-like reactions using mutations to the enzyme's adenine-binding pocket, distal to the site of catalysis. Screening single-site saturation mutagenesis libraries identified mutations that increased catalytic efficiency up to 10-fold in 7 out of 10 enzymes. The enzymes improved in this study represent three different cofactor-binding folds (Rossmann, DHQS-like, and FAD/NAD binding) and utilize both NADH and NADPH. Structural and biochemical analyses show that the improved activities are accompanied by minimal changes in other properties (cooperativity, thermostability, pH optimum, uncoupling), and initial tests on two enzymes (ScADH6 and EcFucO) show improved functionality in Escherichia coli.