Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution
Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution
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通过实验室进化创造出具有高活性和改变催化机制的人工半胱氨酸脂肪酶
DOI：
10.1038/s41467-019-11155-3
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发表时间：
2019-07-19
影响因子：
16.6
通讯作者：
Reetz, Manfred T.
中科院分区：
文献类型：
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作者：
Cen, Yixin;Singh, Warispreet;Reetz, Manfred T.
设计具有高活性和不同于天然酶的催化机制的人工酶是蛋白质设计中的一个挑战。例如，许多人试图通过在各自的催化三元组中引入Ser -> Cys交换来获得活性水解酶，但这通常会导致活性的破坏。我们现在报告说，如果定向进化引入了额外的突变，这个长期存在的教条不再适用。以南极假丝酵母脂肪酶B （CALB）为模型酶，以Ser-His-Asp催化三元组为模式酶，可进化出具有Cys-His-Asp催化三元组和附加突变W104V/A281Y/A282Y/V149G的高活性半胱氨酸脂肪酶，其水解4-硝基苯苯甲酸酯的催化效率比野生型CALB高40倍，并能耐受大体积底物。晶体结构，动力学，MD模拟和QM/MM计算揭示了动力学特征并解释了所有结果，包括涉及两性离子对Cys105(-)/His224（+）的两步机制的偏好，而不是协调过程。
Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser -> Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105(-)/His224(+) rather than a concerted process.