通过理性结合口袋工程来增强3 - 基基甲基酸酶Tfu_0875的活性和基尼-CoA特异性
Lixia Liu1,2, Shuang Liu1,2, Xiangyang Hu1,2
1National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.
Synthetic and systems biotechnology
|May 2, 2024
概括
改造的3 - 基-CoA硫酶Tfu_0875显示了对基尼-CoA的增强活性和特异性,促进了酸的产生. 计算蛋白质工程策略改善了生物基化学合成的酶性能.
科学领域:
- 生物化学和结构生物学
- 酶工程是什么? 酶工程是什么?
- 代谢工程是代谢工程.
背景情况:
- 3-基-CoA硫酶对于线性二碳酸生产至关重要,但其基底特异性和催化效率有限.
- 由于现有的酶的性能不足,酸的产生受到阻碍.
研究的目的:
- 为了从生物化学和结构上分析高效的3 - 基基-CoA硫酶,Tfu_0875.5.
- 为了增强Tfu_0875的活性和特异性,用于基尼尔-CoA,这是基酸的关键前体.
- 为了改进酸生产,设计一种优质的酶变体.
主要方法:
- 野生类型的生物化学和结构分析 Tfu_0875.5.
- 深度学习 (DLKcat) 用于突变识别和贪的蛋白质工程积累策略 (GRAPE) 用于突变积累.
- 位点定向的突变发生和酶变体的表征.
主要成果:
- 改造的突变Tfu_0875N249W/L163H/E217L表现出320%的特异活性和增强的糖-CoA特异性 (Km=0.59毫米).
- 突变者由于酶-基质相互作用的增加和精细的结合口袋,显著减少了基质结合能量 (-30.25 kcal mol-1).
- 酸标位在摇瓶实验中使用工程酶增加了1.35倍.
结论:
- 设计的Tfu_0875N249W/L163H/E217L是一种高效的变种,用于酸-CoA的转化.
- 酶工程策略,包括计算方法,可以有效地改善工业应用中的酶性能.
- 这项工作为酸生产提供了一个有前途的酶,以及一个可转移的策略,用于为生物基化学合成设计其他硫酸酶.
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