醇脱氢酶金属离子绑定位点的可替换性  

Substitutability of metal-binding sites in an alcohol dehydrogenase

作　　者：毕悦欣 蒋迎迎 覃宗敏 曲戈 孙周通 BI Yuexin;JIANG Yingying;QIN Zongmin;QU Ge;SUN Zhoutong(School of Life Sciences,University of Science and Technology of China,Hefei 230026,Anhui,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;University of Chinese Academy of Sciences,Beijing 100049,China;National Technology Innovation Center of Synthetic Biology,Tianjin 300308,China)

机构地区：[1]中国科学技术大学生命科学学院,安徽合肥230026 [2]中国科学院天津工业生物技术研究所,天津300308 [3]中国科学院大学,北京100049 [4]国家合成生物技术创新中心,天津300308

出　　处：《生物工程学报》2022年第4期1518-1526,共9页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2019YFA0905100);天津市自然科学基金(19JCQNJC09100);天津市合成生物技术创新能力提升行动项目(TSBICIP-CXRC-009)。

摘　　要：金属酶通过其极性氨基酸残基侧链所形成的共价键去锚定金属离子,目前鲜有报道替换金属绑定位点本身是否影响原有酶催化性能。以来源于Thermoanaerobacter brockii的锌离子依赖型醇脱氢酶TbSADH为研究对象,对其绑定锌离子的3个氨基酸残基位点Cys37、His59及Asp150进行序列保守性分析并构建突变体文库。经过建库筛选,从224个克隆中获得3个能够不对称催化还原模式底物四氢呋喃-3-酮的突变体,且转化率及ee值与野生型酶保持一致。结果揭示了虽然醇脱氢酶TbSADH的金属离子绑定位点高度保守,但仍具有一定的可替换性,且不以损耗活性及立体选择性为代价,为后续金属离子置换及其介导的新反应设计奠定了研究基础。Covalently anchoring of a ligand/metal via polar amino acid side chain(s)is often observed in metalloenzyme,while the substitutability of metal-binding sites remains elusive.In this study,we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii(TbSADH)as a model enzyme,analyzed the sequence conservation of the three residues Cys37,His59,and Asp150 that bind the zinc ion,and constructed the mutant library.After experimental validation,three out of 224 clones,which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one,were screened out.The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity,which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.

关 键 词：酶工程 生物催化 理性设计 金属酶 金属绑定位点 

分 类 号：Q814[生物学—生物工程]