Precise regulation of the substrate selectivity of Baeyer-Villiger monooxygenase to minimize overoxidation of prazole sulfoxides

doi:10.1016/S1872-2067(23)64482-1

Abstract

Abstract:

Baeyer-Villiger monooxygenases (BVMOs) can catalyze the asymmetric oxidation of sulfides to valuable chiral sulfoxides, but the overoxidation of sulfoxides to undesired sulfones limits the synthetic application of BVMOs. This overoxidation is caused by insufficient substrate selectivity of BVMOs, where the desired product sulfoxide can be further oxidized. In this study, a mathematical model was constructed to quantitatively define the substrate selectivity based on the ratio of the specificity constant (k_cat/K_m) between sulfide and sulfoxide. The substrate selectivity of a pyrmetazole monooxygenase (AcPSMO) was precisely regulated using a structure-guided substrate tunnel engineering approach, which successfully minimized sulfoxide overoxidation. The sulfone content of variant F277L was less than 1% (mol/mol), compared with 65% for the wild-type, in the pyrmetazole oxidation reaction after 24 h. Molecular dynamics simulations and quantum mechanics/molecular mechanics studies showed that the altered H-bonding networks surrounding the flavin hydroperoxide (FADH-OOH) can modulate the mechanism and activity for sulfoxide oxidation. Furthermore, the redesigned mutants of AcPSMO were successfully applied for the controllable synthesis of other chiral prazole sulfoxides.

Key words: Baeyer-Villiger monooxygenase, Chiral sulfoxide, Overoxidation, Substrate selectivity, Protein engineering, Molecular dynamics simulation, Quantum mechanics/molecular, mechanics study

Yinqi Wu, Qianqian Chen, Qi Chen, Qiang Geng, Qiaoyu Zhang, Yu-Cong Zheng, Chen Zhao, Yan Zhang, Jiahai Zhou, Binju Wang, Jian-He Xu, Hui-Lei Yu. Precise regulation of the substrate selectivity of Baeyer-Villiger monooxygenase to minimize overoxidation of prazole sulfoxides[J]. Chinese Journal of Catalysis, 2023, 51: 157-167.

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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(23)64482-1

https://www.cjcatal.com/EN/Y2023/V51/I8/157

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Enzyme	c^a (%)	ee ^b (S, %)	pp^a (%)	R_app^c
WT	98.7 ± 0.7	99.1	76.6 ± 1.8	12.6
L244V	62.1 ± 1.0	97.2	97.8 ± 0.2	25.2
F277V	93.2 ± 2.4	99.0	98.9 ± 0.2	170
F281V	95.1 ± 1.5	98.0	89.8 ± 1.1	20.1
L432V	83.8 ± 0.4	92.2	47.8 ± 0.7	1.44
A433V	97.7 ± 0.8	99.1	92.3 ±1.0	37.1

Enzyme	c^a (%)	ee ^b (S, %)	pp^a (%)	R_app^c
WT	98.7 ± 0.7	99.1	76.6 ± 1.8	12.6
L244V	62.1 ± 1.0	97.2	97.8 ± 0.2	25.2
F277V	93.2 ± 2.4	99.0	98.9 ± 0.2	170
F281V	95.1 ± 1.5	98.0	89.8 ± 1.1	20.1
L432V	83.8 ± 0.4	92.2	47.8 ± 0.7	1.44
A433V	97.7 ± 0.8	99.1	92.3 ±1.0	37.1

Enzyme	Pyrmetazole			(S)-Omeprazole
Enzyme	k_cat (min^‒1)	K_m (mmol/L)	k_cat/K_m (L min^‒1 mmol^‒1)	k_cat (min^‒1)	K_m (mmol L^‒1)	k_cat/K_m (L min^‒1 mmol^‒1)
L432V	0.42 ± 0.01	0.10 ± 0.01	4.39	3.77 ± 0.37	0.38 ± 0.07	9.93
WT	0.30 ± 0.01	0.20 ± 0.02	1.51	0.64 ± 0.01	0.85 ± 0.19	0.75
F277L	0.74 ± 0.09	0.14 ± 0.02	5.31	0.08 ± 0.00	1.70 ± 0.09	0.05

Enzyme	Pyrmetazole			(S)-Omeprazole
Enzyme	k_cat (min^‒1)	K_m (mmol/L)	k_cat/K_m (L min^‒1 mmol^‒1)	k_cat (min^‒1)	K_m (mmol L^‒1)	k_cat/K_m (L min^‒1 mmol^‒1)
L432V	0.42 ± 0.01	0.10 ± 0.01	4.39	3.77 ± 0.37	0.38 ± 0.07	9.93
WT	0.30 ± 0.01	0.20 ± 0.02	1.51	0.64 ± 0.01	0.85 ± 0.19	0.75
F277L	0.74 ± 0.09	0.14 ± 0.02	5.31	0.08 ± 0.00	1.70 ± 0.09	0.05

Substrate	Enzyme	c^a (%, 12 h)	pp^a (%)	R_app^b
Pymetazole	WT	51.9 ± 1.2	97.9 ± 0.1	78.3
	F277L	96.7 ± 0.6	98.9 ± 0.1	266
	D57Q	55.4 ± 1.1	98.7 ± 0.1	126
	F277L/D57Q	94.6 ± 0.1	99.1 ± 0.0	321
Lansoprazole sulfide	WT	58.7 ± 4.6	78.2 ± 1.7	7.53
	F277L	99.1 ± 0.0	97.2 ± 0.4	163
	F277L/D57Q	69.4 ± 1.7	97.6 ± 0.1	68.1
Pantoprazole sulfide	WT	99.9 ± 0.0	35.6 ± 1.1	7.36
	F277L	99.8 ± 0.0	95.3 ± 0.3	124
	F277L/D57Q	99.8 ± 0.0	96.7 ± 0.0	186
Ilaprazole sulfide	WT	75.2 ± 1.0	16.5 ± 0.4	2.00
	F277L	86.5 ± 0.1	93.1 ± 0.2	30.5
	F277L/D57Q	71.1 ± 0.4	99.2 ± 0.0	203