Catalytic Promiscuity-driven Redesign of Enzyme Functions

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

Abstract

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

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

Figures/Tables 6

Fig. 1 Schematic representation of evolutionary enzyme catalytic promiscuity

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

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]

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）

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

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

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