School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, China.
Department of Cardiology, Affiliated Hospital of Jiangnan University, Wuxi, China.
Appl Environ Microbiol. 2023 May 31;89(5):e0010923. doi: 10.1128/aem.00109-23. Epub 2023 Apr 18.
d--hydroxyphenylglycine (d-HPG) is an important intermediate in the pharmaceutical industry. In this study, a tri-enzyme cascade for the production of d-HPG from l-HPG was designed. However, the amination activity of Prevotella timonensis -diaminopimelate dehydrogenase (DAPDH) toward 4-hydroxyphenylglyoxylate (HPGA) was identified as the rate-limiting step. To overcome this issue, the crystal structure of DAPDH was solved, and a "binding pocket and conformation remodeling" strategy was developed to improve the catalytic activity toward HPGA. The best variant obtained, DAPDH, exhibited a catalytic efficiency (/) that was 26.75-fold higher than that of the wild type. This improvement was due to the enlarged substrate-binding pocket and enhanced hydrogen bond networks around the active center; meanwhile, the increased number of interdomain residue interactions drove the conformation distribution toward the closed state. Under optimal transformation conditions, DAPDH produced 19.8 g/L d-HPG from 40 g/L racemate DL-HPG in a 3 L fermenter within 10 h, with 49.5% conversion and >99% enantiomeric excess. Our study provides an efficient three-enzyme cascade pathway for the industrial production of d-HPG from racemate DL-HPG. d--hydroxyphenylglycine (d-HPG) is an important intermediate in the synthesis of antimicrobial compounds. d-HPG is mainly produced via chemical and enzymatic approaches, and enzymatic asymmetric amination employing diaminopimelate dehydrogenase (DAPDH) is considered an attractive method. However, the low catalytic activity of DAPDH toward bulky 2-keto acids limits its applications. In this study, we identified a DAPDH from Prevotella timonensis and created a mutant, DAPDH, which exhibited a catalytic efficiency (/) toward 4-hydroxyphenylglyoxylate that was 26.75-fold higher than that of the wild type. The novel strategy developed in this study has practical value for the production of d-HPG from inexpensive racemate DL-HPG.
d--羟基苯甘氨酸（d-HPG）是医药行业的重要中间体。本研究设计了一种从 l-HPG 生产 d-HPG 的三酶级联反应。然而，Prevotella timonensis -二氨基庚二酸脱氢酶（DAPDH）对 4-羟基苯甘氨酸（HPGA）的胺化活性被鉴定为限速步骤。为了克服这个问题，我们解析了 DAPDH 的晶体结构，并开发了一种“结合口袋和构象重塑”策略来提高其对 HPGA 的催化活性。获得的最佳变体 DAPDH 的催化效率（/）比野生型高 26.75 倍。这种改进归因于扩大的底物结合口袋和增强的活性中心周围氢键网络；同时，增加的结构域间残基相互作用促使构象分布向封闭状态移动。在最佳转化条件下，DAPDH 在 3 L 发酵罐中 10 小时内从 40 g/L 外消旋 DL-HPG 生产 19.8 g/L d-HPG，转化率为 49.5%，对映体过量值>99%。本研究为从外消旋 DL-HPG 工业生产 d-HPG 提供了一种高效的三酶级联途径。
d--羟基苯甘氨酸（d-HPG）是合成抗菌化合物的重要中间体。d-HPG 主要通过化学和酶法生产，使用二氨基庚二酸脱氢酶（DAPDH）的酶促不对称胺化被认为是一种有吸引力的方法。然而，DAPDH 对大体积 2-酮酸的低催化活性限制了其应用。在这项研究中，我们鉴定了一种来自 Prevotella timonensis 的 DAPDH，并创建了一个突变体 DAPDH，它对 4-羟基苯甘氨酸的催化效率（/）比野生型高 26.75 倍。本研究开发的新策略对于从廉价的外消旋 DL-HPG 生产 d-HPG 具有实际价值。
