P450cin介导电子转移的定向进化

Ketaki D. Belsare, Thomas Horn, A. J. Ruff, Ronny Martínez, Anders O. Magnusson, D. Holtmann, J. Schrader, U. Schwaneberg
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引用次数: 16

摘要

定向进化是优化酶性能以满足应用需求的一种有效方法。有趣的目标是P450单加氧酶,它催化化学惰性碳氢键的立体和区域特异性羟基化。p450在无细胞反应条件下的合成受到总周转率低、酶不稳定、产物产率低以及需要昂贵的辅助因子NADPH的限制。生物电催化是在无细胞p450催化反应中替代NADPH的一种替代方法。然而,天然酶通常不适合使用非天然的电子传递系统。在这里,我们报告了先前设计的P450 CinA-10aa-CinC融合蛋白(命名为p450cina - add - cinc)的定向进化,以锌/钴(III)墓状物作为电子传递系统,增加1,8-桉树脑的羟基化活性。两轮序列饱和突变(SeSaM)之后,P450 china -10aa- cinc融合蛋白进行一轮多位点饱和突变,产生一个变体(Gln385His, Val386Ser, Thr77Asn, Leu88Arg;命名为KB8),催化效率(28µM−1 min−1)比P450cin-ADD-CinC(7µM−1 min−1)提高3.8倍。此外,变体KB8的生成量(500µMµM−1 P450)比使用NADPH作为辅助因子(315µMµM−1 P450)的等摩尔china, CinC和Fpr的混合物高1.5倍。此外,以铂/钴(III)墓墓为电子传递体系的电化学实验表明,KB8变体的产物形成率(0.16 nmol (nmol) P450−1 min−1 cm−2)比P450cin-ADD-CinC (0.04 nmol (nmol) P450−1 min−1 cm−2)高4倍。总之,目前的工作显示了使用定向进化生成适合用于替代电子传递系统的P450酶的前景。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Directed evolution of P450cin for mediated electron transfer
Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C–H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions. However, natural enzymes are often not suitable for using non-natural electron delivery systems. Here we report the directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III)sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of Sequence Saturation Mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generated a variant (Gln385His, Val386Ser, Thr77Asn, Leu88Arg; named KB8) with a 3.8-fold increase in catalytic efficiency (28 µM−1 min−1) compared to P450cin-ADD-CinC (7 µM−1 min−1). Furthermore, variant KB8 exhibited a 1.5-fold higher product formation (500 µM µM−1 P450) compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as co-factor (315 µM µM−1 P450). In addition, electrochemical experiments with the electron delivery system platinum/cobalt(III)sepulchrate showed that the KB8 variant had a 4-fold higher product formation rate (0.16 nmol (nmol) P450−1 min−1 cm−2) than the P450cin-ADD-CinC (0.04 nmol (nmol) P450−1 min−1 cm−2). In summary, the current work shows prospects of using directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems.
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