Cahn J K B, Baumschlager A, Brinkmann-Chen S, Arnold F H
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E California Blvd, MC 210-41, Pasadena, CA 91125, USA.
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E California Blvd, MC 210-41, Pasadena, CA 91125, USA
Protein Eng Des Sel. 2016 Jan;29(1):31-8. doi: 10.1093/protein/gzv057. Epub 2015 Oct 27.
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.
NAD(P)H 依赖性酶在新陈代谢和细胞过程中普遍存在，并且在制药和工业应用中也备受关注。在此，我们提出一种基于结构的酶工程策略，通过对酶的腺嘌呤结合口袋（位于催化位点远端）进行突变，来改善 NAD(P)H 依赖性酶对天然或类似天然反应的催化特性。筛选单位点饱和诱变文库确定了 10 种酶中的 7 种酶的突变，这些突变使催化效率提高了 10 倍。本研究中得到改进的酶代表三种不同的辅因子结合折叠方式（Rossmann、DHQS 样和 FAD/NAD 结合），并且同时利用 NADH 和 NADPH。结构和生化分析表明，活性的提高伴随着其他性质（协同性、热稳定性、最适 pH、解偶联）的最小变化，对两种酶（ScADH6 和 EcFucO）的初步测试表明在大肠杆菌中功能得到改善。
