基于极性扫描的L-苏氨酸醛缩酶β-碳立体选择性分子机制研究

批准号:
32000898
项目类别:
青年科学基金项目
资助金额:
24.0 万元
负责人:
蔡雪
依托单位:
学科分类:
蛋白质、多肽与酶生物化学
结题年份:
2023
批准年份:
2020
项目状态:
已结题
项目参与者:
蔡雪
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中文摘要
L-苏氨酸醛缩酶(L-TA)能够可逆催化不同醛与α-甘氨酸的羟醛缩合反应,是有机合成重要手性砌块β-羟基-α-氨基酸的关键酶,但该酶的β-碳立体选择性较差,限制其在手性医药中间体合成中的应用。前期工作中,我们筛选了4种不同来源的L-TA,并对其酶学特性进行了系统表征。在此基础上,本项目拟以筛选的一种立体选择性较高的L-TA为研究对象,首先以天冬酰胺和缬氨酸分别作为极性和非极性筛子,极性扫描突变该酶底物结合口袋与β-碳立体选择性相关的位点;随后,迭代饱和突变已验证的正向突变位点,获得β-碳位高立体选择性的L-TA,并对其晶体结构进行解析;最终,利用预反应态模型和约束条件下的分子动力学模拟技术,系统分析正/反向突变在位点/类型、结合口袋形状/极性、反应动力学上的差异,阐明L-TA的β-碳立体选择性分子机制,为获得具有工业化应用前景的L-TA奠定基础。
英文摘要
L-threonine aldolase catalyzes the reversible aldol condensation reaction between aldehydes and α-glycine. It is a key enzyme for the organic synthesis of important chiral building blocks β-hydroxy-α-amino acid, but the poor stereoselectivity of β-carbon limits its application in the synthesis of chiral pharmaceutical intermediates. Previously, we screened four different sources of L-TA and characterized their enzymatic properties systematically. On this basis, a previously isolated L-TA with relatively high stereoselectivity will be used in this project. Firstly, asparagine and valine are used as polar and non-polar sieves in the polarity scanning strategy to mine the amino acids in the substrate binding pocket relevant with β-carbon stereoselectivity; then, iterative saturation mutagenesis on the confirmed positive mutation sites will be conducted to construct mutant L-TA with high β-carbon stereoselectivity and its crystal structure will be analyzed; finally, prereaction-state model and molecular dynamics simulation under constrained conditions will be applied to systematically analyze the differences in positive/nagative mutations at the site/type, shape/polarity of the binding pocket, kinetics in order to clarify the molecular mechanism of β-carbon stereoselectivity in L-TA and lay the foundation for obtaining L-TA with industrial application prospects.
期刊论文列表
专著列表
科研奖励列表
会议论文列表
专利列表
DOI:10.1021/acssuschemeng.3c02691
发表时间:2023-06
期刊:ACS Sustainable Chemistry & Engineering
影响因子:--
作者:Xue Cai;Jing Hua;Zhengyou Lin;Chen-Yang Sun;Chang-Hui Hu;Xiao‐Jian Zhang;Ji-dong Shen;Hai-yan Zhou;Hong-Yan Wang;Kai-Qian Chen;De-Shui Chen;Xin-Ping Cheng;Mian Li;Zhi-qiang Liu;Yuguo Zheng
通讯作者:Xue Cai;Jing Hua;Zhengyou Lin;Chen-Yang Sun;Chang-Hui Hu;Xiao‐Jian Zhang;Ji-dong Shen;Hai-yan Zhou;Hong-Yan Wang;Kai-Qian Chen;De-Shui Chen;Xin-Ping Cheng;Mian Li;Zhi-qiang Liu;Yuguo Zheng
DOI:10.1016/j.eng.2022.03.020
发表时间:2022-10
期刊:Engineering
影响因子:12.8
作者:Xue Cai;Jiang-Wei Shen;Yu Qiang;Jing Hua;Zhang-Qi Ma;Zhi-qiang Liu;Yuguo Zheng
通讯作者:Xue Cai;Jiang-Wei Shen;Yu Qiang;Jing Hua;Zhang-Qi Ma;Zhi-qiang Liu;Yuguo Zheng
DOI:10.1007/s00449-021-02611-w
发表时间:2021-07-15
期刊:BIOPROCESS AND BIOSYSTEMS ENGINEERING
影响因子:3.8
作者:Cai, Xue;Jin, Jie-Yi;Zheng, Yu-Guo
通讯作者:Zheng, Yu-Guo
基于廉价碳源的维生素B5生物合成途径的重构及调控机制研究
- 批准号:HDMZ25C050001
- 项目类别:省市级项目
- 资助金额:0.0万元
- 批准年份:2025
- 负责人:蔡雪
- 依托单位:
L-苏氨酸醛缩酶有机溶剂耐受性的构效机理研究
- 批准号:LQ21B060006
- 项目类别:省市级项目
- 资助金额:0.0万元
- 批准年份:2020
- 负责人:蔡雪
- 依托单位:
国内基金
海外基金
