Protein Engineering for Enhanced Acyltransferase Activity, Substrate Scope, and Selectivity of the Mycobacterium smegmatis Acyltransferase MsAcT
Protein Engineering for Enhanced Acyltransferase Activity, Substrate Scope, and Selectivity of the Mycobacterium smegmatis Acyltransferase MsAcT
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DOI：
10.1021/acscatal.0c01767
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发表时间：
2020-07-17
期刊：
影响因子：
12.9
通讯作者：
Bornscheuer, Uwe T.
中科院分区：
文献类型：
--
作者：
Godehard, Simon P.;Badenhorst, Christoffel P. S.;Bornscheuer, Uwe T.
来自耻垢分枝杆菌的高效且多功能的酰基转移酶 MsAcT 可催化水性酰基转移反应，使其能够在环保过程和酶级联中应用。我们合理地设计了几种变体，将酰基转移与水解的比率提高了 30 倍，同时大部分保留了初始活性。还设计了表现出更广泛的酰基供体底物范围和更高或反向对映选择性的变体。催化组氨酸-天冬氨酸对的改变降低了水解水的活化，从而增加了酰基转移与水解的比率。这项研究表明，针对水网络的破坏和操纵亲核试剂的激活是工程混杂酰基转移酶活性的有前途的策略。
The highly efficient and versatile acyltransferase MsAcT from Mycobacterium smegmatis catalyzes aqueous acyl transfer reactions, enabling applications in environmentally friendly processes and enzyme cascades. We rationally designed several variants with up to 30-fold increased acyl transfer to hydrolysis ratios while mostly retaining initial activity. Variants exhibiting broader acyl-donor substrate scope and higher or inverted enantioselectivity were also designed. Alterations of the catalytic His-Asp pair decreased the activation of hydrolytic water, thereby increasing acyl transfer to hydrolysis ratios. This study demonstrates that targeting the disruption of water networks and manipulating the activation of nucleophiles are promising strategies for engineering promiscuous acyltransferase activities.