Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot
Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot
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DOI：
10.1002/cbic.201500529
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发表时间：
2015-12
期刊：
影响因子：
3.2
通讯作者：
Cindy Wechsler;Danilo Meyer;S. Loschonsky;L. Funk;P. Neumann;R. Ficner;Florian Brodhun;Michael Müller;K. Tittmann
中科院分区：
文献类型：
--
作者：
Cindy Wechsler;Danilo Meyer;S. Loschonsky;L. Funk;P. Neumann;R. Ficner;Florian Brodhun;Michael Müller;K. Tittmann
对映体选择性键的建立和断裂是酶作用的一个标志，然而改变反应的对映体选择性是一项困难的工作，通常需要对突变库和多个突变进行广泛的筛选。在这里，我们证明了丙酮酸脱羧酶中单一催化热点的突变多样化使得乙酰胆碱和苯乙酰甲醇的对映体都可以获得，在乙酰胆碱的情况下，甚至从非选择性的野生型蛋白开始。蛋白质结晶学被用来使这些发现合理化，并提出了一个如何控制对映体选择性的机制模型。在更广泛的背景下，我们的研究强调了机制启发和结构指导的合理蛋白质设计通过系统地探索单个热点的生物催化潜力来提高和切换酶反应的对映选择性的效率。
Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild‐type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a broader context, our studies highlight the efficiency of mechanism‐inspired and structure‐guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions, by systematically exploring the biocatalytic potential of a single hot spot.