酶法合成(S)-3-羟基四氢呋喃  

Enzymatic synthesis of(S)-3-hydroxytetrahydrofuran

作　　者：李润通 李旭 袁波 郭敬功[1] 曲戈 孙周通 LI Runtong;LI Xu;YUAN Bo;GUO Jinggong;QU Ge;SUN Zhoutong(School of Life Sciences,Henan University,Kaifeng 475000,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;National Center of Technology Innovation for Synthetic Biology,Tianjin 300308,China;Key Laboratory of Engineering Biology for Low-Carbon Manufacturing,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)

机构地区：[1]河南大学生命科学学院,开封475000 [2]中国科学院天津工业生物技术研究所,天津300308 [3]国家合成生物技术创新中心,天津300308 [4]中国科学院天津工业生物技术研究所低碳合成工程生物学重点实验室,天津300308

出　　处：《生物学杂志》2025年第2期1-6,51,共7页Journal of Biology

基　　金：天津市合成生物技术创新能力提升行动项目(TSBICIP-CXRC-053)。

摘　　要：(S)-3-羟基四氢呋喃是一种重要的医药中间体,其化学法合成面临手性控制不好、成本高等挑战,作为替代方案,醇脱氢酶可一步催化底物二氢-3(2H)-呋喃酮的不对称还原反应,生成相应的醇产物。研究通过筛选醇脱氢酶库,获得来源于Aromatoleum aromaticum EbN1的醇脱氢酶PED,能够不对称催化还原二氢-3(2H)-呋喃酮合成(S)-3-羟基四氢呋喃。通过反应条件优化,PED催化该反应的转化率>99%,催化效率(k cat/K_(m))为0.40 L/(mmol·s),产物ee值为90%,且维持较好的热稳定性(T_(m)=67℃)。通过计算分析酶关键残基与底物的相互作用,揭示了PED催化底物还原的立体选择性控制机制,即S141、Y151和Y154关键残基与底物形成氢键相互作用网络,促进形成(S)-前手性构象,辅酶NADH中氢负离子转移至羰基碳原子,将底物还原为(S)-产物。此外,放大反应实验表明PED可耐受400 mmol/L底物浓度,并可在5 h内实现完全转化。通过分离纯化,产物收率为74.7%,ee值为90%。研究挖掘到一个新颖醇脱氢酶PED,并对其酶学性能进行表征,拓展了生物法合成(S)-3-羟基四氢呋喃的酶工具箱。(S)-3-Hydroxytetrahydrofuran is an important pharmaceutical intermediate,and its chemical synthesis meets challenges such as low stereoselectivity and high cost.Alternatively,alcohol dehydrogenases catalyze the one-step asymmetric reduction of dihydro-3(2H)-furanone to the corresponding alcohol product.In this study,after screening an alcohol dehydrogenase library,an alcohol dehydrogenase derived from Aromatoleum aromaticum EbN1(PED)was obtained,which enabled the reduction of dihydro-3(2H)-furanone to(S)-3-hydroxytetrahydrofuran.By optimizing the reaction conditions,PED catalyzed the model reaction with a>99%conversion.The catalytic efficiency(k cat/K_(m))was measured at 0.40 L/(mmol·s),with an ee value of 90%.Additionally,PED demonstrated a good thermostability,maintaining a T_(m)value of 67℃.Computational analysis revealed the mechanism of stereoselectivity in the reduction of substrates catalyzed by PED.The key residues such as S141,Y151 and Y154 formed a network of hydrogen-bonding interactions with the substrate,which promoted the formation of a pro-(S)conformation of the substrate.The(S)-product was then formed by the migration of a hydride from the coenzyme NADH to the carbonyl carbon of the substrate.Furthermore,large-scale reactions showed that PED can completely convert the substrate concentration up to 400 mmol/L in 5 h,with an isolated yield of 74.7%and an ee value of 90%.In this study,a novel alcohol dehydrogenase PED was identified and characterized with enzymatic performance,expanding the repertoire of enzymes available for the biocatalysis of(S)-3-hydroxytetrahydrofuran.

关 键 词：醇脱氢酶 生物催化 不对称催化还原 (S)-3-羟基四氢呋喃 辅酶再生 

分 类 号：Q814[生物学—生物工程]