催化混杂性驱动的酶功能重塑

doi:10.13560/j.cnki.biotech.bull.1985.2022-1305

摘要/Abstract

摘要：

作为生物催化剂，酶蛋白介导的生化反应具有条件温和、绿色环保等优点。然而相比化学催化剂，天然酶功能的局限性制约了它在生物制造领域的广泛应用。前期研究表明，酶蛋白除了催化专一性外，同时还展现出混杂性的一面，可在特定条件下催化非天然模式反应。这一特性为酶分子功能重塑提供了新思路，可用来指导人工酶设计，拓展天然酶的催化边界，实现新颖酶促反应类型，以扩大酶催化应用场景。本文从酶催化功能混杂性背后可能的进化机制入手，综述了当前诱导酶催化功能混杂性的常用策略，如定向进化、构象动力学、反应条件诱导及祖先酶重构等技术，并从催化机制、构效关系及适应性进化等多个角度，结合近年来相关研究实例，探讨了催化功能混杂性背后的分子机制，为突破天然酶促反应局限性、创制催化非天然反应的高效人工酶元件提供参考。

关键词: 催化混杂性, 定向进化, 理性设计, 酶工程, 生物催化

Abstract:

As biocatalysts, the reactions directed by enzymes can be conducted in a green and sustainable fashion under mild conditions. However, compared to the conversional chemical catalysts, functional limitations of native enzymes restrict their broad applications in biomanufacturing. Previous studies reveal that enzymes also have catalytic promiscuities in addition to catalytic specificity, which are able to catalyze non-natural reactions. This property sheds light on the enzyme redesign, which can be used to guide the design of artificial enzymes, expand the catalytic boundaries of natural enzymes, achieve novel enzymatic reaction types, and broaden the application scenarios of enzyme catalysis. Based on the evolutionary mechanism of catalytic promiscuity, this review summarized the common strategies used for inducing promiscuities, including directed evolution, conformational dynamics, manipulating reaction conditions, ancestor enzyme reconstruction. This review also explored the molecular mechanism behind the catalytic promiscuity in the view of catalytic mechanism, structure-function relationship and adaptive evolution combined with recent relevant study cases. It may provide a reference for breaking through the limitations of natural enzymatic reactions and creating efficient artificial enzyme components that catalyze unnatural reactions.

Key words: catalytic promiscuity, directed evolution, rational design, enzyme engineering, biocatalysis

曲戈, 孙周通. 催化混杂性驱动的酶功能重塑[J]. 生物技术通报, 2023, 39(4): 1-9.

QU Ge, SUN Zhou-tong. Catalytic Promiscuity-driven Redesign of Enzyme Functions[J]. Biotechnology Bulletin, 2023, 39(4): 1-9.

图/表 6

图1 酶催化功能混杂性进化示意图

Fig. 1 Schematic representation of evolutionary enzyme catalytic promiscuity

图2 人工Kemp消除酶设计 a：Kemp消除反应方程式；b：基于不同模板获得的KE07、KE59及KE70，其中KE59以KE59 R1 7/10H突变体结构展示；c：基于木聚糖酶设计的HG3

Fig. 2 Design of artificial Kemp elimination enzymes a: Formula of Kemp elimination reaction. b: KE07, KE59 and KE70 designed from three distinct templates. KE59 is depicted using the crystallographic structure of its mutant R1 7/10H. c: HG3 designed from a xylanase

表1 人工Kemp消除酶相关代表性突变体列表

Table 1 Representative mutants related to artificial Kemp elimination enzymes

Template	Enzyme	k_cat(s^-1)	K_m(mM^-1)	k_cat/K_m(M^-1s^-1)	Reference
裂合酶等	KE07	0.018	1.4	12.2	[17]
	KE59	0.29	1.8	163	[17]
	KE70	0.16	2.1	78.3	[17]
	KE07.7	1.37	0.54	2 590	[18]
	KE59.13	9.53	0.16	60 430	[19]
	KE70.6	5.0	0.088	57 300	[20]
木聚糖酶	HG3	0.68	1.6	425.0	[21]
	HG3.17	700	3.0	230 000	[22]
P450 BM3	P450-BM3	1.5	6	240	[23]
	A82F	8.4	0.27	31 000	[23]

图3 构象动力学指导的TbSADH酶改造 a：催化反应示意图；b：底物结合口袋关键位点的运动性分析；c-d：A85及I86两位点突变后（c）与突变前（d）所在loop区域柔性分析；e：关键位点P84的空间位置；f-h：野生型（f）与两个最优突变体ΔP84/A85G（g）及P84S/I86L（h）的构象自由能路径分布

Fig. 3 Conformational dynamics-guided modification of enzyme TbSADH a: Schematic representation of catalyzed reaction. b: Dynamics analysis of the substrate binding pocket. c-d: Flexibility exploration of the loop region that accommodates residues A85 and I86 after（c）and before mutagenesis（d）. e: Position of residue P84. f-h: Free energy landscapes（kcal/mol）of wild type（f）, ΔP84/A85G（g）and P84S/I86L（h）

图4 羧酸还原酶介导的混杂性催化反应类型 a：氧化还原反应；b：分子间酰胺化反应；c：分子间酯化反应；d：分子内酰胺化反应

Fig. 4 Promiscuous reactions mediated by carboxylic acid reductase a: Redox reaction; b: intermolecular amidation; c: intermolecular esterification; d: intramolecular lactamization

图5 环己二烯脱水酶（a）与查尔酮异构酶（b）的祖先序列重构

Fig. 5 Ancestral sequence reconstruction of cyclohexadienyl dehydratase（a）and chalcone isomerase（b）

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