Department of Chemistry, Zhejiang University, 310027, Hangzhou, China.
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 200032, Shanghai, China.
Nat Commun. 2019 Jul 19;10(1):3198. doi: 10.1038/s41467-019-11155-3.
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.
利用工程学手段设计出具有与天然酶不同的活性和催化机制的人工酶是蛋白质设计领域的一项挑战。例如，人们曾尝试通过在相应的催化三联体上引入 Ser ⁇ Cys 取代来获得具有活性的水解酶，但这通常会导致酶失活。我们现在报告称，只要通过定向进化引入额外的突变，这一长期以来的教条就不再适用。我们选用南极假丝酵母脂肪酶 B（CALB）作为具有 Ser-His-Asp 催化三联体的模型酶，通过定向进化获得了一个具有 Cys-His-Asp 催化三联体和额外突变 W104V/A281Y/A282Y/V149G 的高活性半胱氨酸脂肪酶，其对 4-硝基苯苯甲酸的水解催化效率比野生型 CALB 高 40 倍，并且能够耐受大体积的底物。晶体结构、动力学、MD 模拟和 QM/MM 计算揭示了动态特征，并解释了所有结果，包括对涉及两性离子对 Cys105/His224 的两步机制的偏好，而不是协同过程。
