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为合成三级γ-基氨基酸的脱氧化合酶的变异性引导进化

Meghan E Campbell¹, Amanda R Ohler¹, Matthew J McGill¹

¹Department of Chemistry, University of Wisconsin-Madison, USA, Madison, WI 53706.

Angewandte Chemie (International ed. in English)

|January 28, 2025

概括

研究人员通过定向进化和基质多重选 (SUMS) 设计了酶 UstD,以实现更广泛的基质活性. 这种方法迅速产生了用于合成非正规氨基酸的多功能酶.

科学领域:

生物催化和酶工程生物催化和酶工程
合成生物学合成生物学
有机化学有机化学

背景情况:

酶应用往往需要在结构多样化的基板上进行活动.
脱碳氧化阿尔多酶 UstD 最初与电友的活性有限,需要工程来改善功能.
定向进化是酶优化的强大工具,但可能导致意想不到的基质特异性.

研究的目的:

用一系列电友来设计 UstD 酶以增强 C-C 键的形成.
开发和应用一个有针对性的进化策略,促进酶普遍性而不是狭窄的特异性.
为了证明基质多重选 (SUMS) 在指导酶进化的有用性.

主要方法:

UstD酶的多代定向进化.
基质多重选 (SUMS) 的应用,以评估不同基质的酶活性.
被确定为与活性部位合的远部残留物的和突变发生.

主要成果:

SUMS有效地引导了 UstD 演变,使其获得更广泛的基质接受.
在活性部位外影响催化功能的突变通过乱交的变化被确定.
工程 UstD 变体在多种基质上表现出互补活性,使非正规氨基酸与三级酒精侧链的合成成为可能.

结论:

开发的方法促进了酶的快速和有效的演变,以实现新的催化功能.
使用SUMS的故意指导通用性是可以实现的,克服了指导进化中的特异性漂移的挑战.
工程 UstD 酶为价值化学化合物的融合合成提供了一个多功能平台.

关键词:

蛋白质工程是一种蛋白质工程.生物催化剂的生物催化剂指导进化是指导进化的.酶氨酸是一种胺.非正规的氨基酸.

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