Engineering of a Baeyer–Villiger monooxygenase reveals key residues for the asymmetric oxidation of omeprazole sulfide

Wei, Shiyu; Xu, Guochao; Lu, Zhou; Zhou, Jieyu; Ni, Ye

doi:10.1039/d2cc04161h

Cited by 5 publications

(3 citation statements)

References 32 publications

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“…The GBRT model demonstrated effectiveness in predicting the enantioselectivity of carboxylesterase AcEst1 towards CHCE. Moreover, the potential application of the GBRT model using the aforementioned features is under evaluation in learning and predicting the enantioselectivity of alcohol dehydrogenase from Kluyveromyces polysporus 58 and Baeyer-Villiger monooxygenase from Rhodococcus aetherivorans 59 .…”

Section: Discussionmentioning

confidence: 99%

Enhanced stereodivergent evolution of carboxylesterase for efficient kinetic resolution of near-symmetric esters through machine learning

Xu,

Dou,

Chen

et al. 2024

Preprint

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Carboxylesterases serve as potent biocatalysts in the enantioselective synthesis of chiral carboxylic acids and esters. However, naturally occurring carboxylesterases exhibit limited enantioselectivity, particularly towards ethyl 3-cyclohexene-1-carboxylate (CHCE), due to its nearly symmetric structure. While machine learning has proven effective in expediting directed evolution, the lack of models for prediction of enantioselectivity for carboxylesterases has hindered progress, primarily due to challenges obtaining high-quality training datasets. In this study, we devised a high-throughput method by coupling alcohol dehydrogenase to determine the apparent enantioselectivity of the carboxylesterase AcEst1 from Acinetobacter sp. JNU9335, thereby generating a high-quality dataset. Leveraging seven features derived from biochemical considerations, we quantitively described the steric, hydrophobic, hydrophilic, electrostatic, hydrogen bonding, and π-π interaction effects of residues within AcEst1. A robust gradient boosting regression tree model was trained to facilitate stereodivergent evolution, resulting in the enhanced enantioselectivity of AcEst1 towards CHCE. Through this approach, we successfully obtained two stereocomplementary variants, DR3 and DS6, demonstrating significantly increased and reversed enantioselectivity. Notably, DR3 and DS6 exhibited utility in the enantioselective hydrolysis of various symmetric esters. Comprehensive kinetic parameter analysis, molecular dynamics simulations, and QM/MM calculations provided insights into the kinetic and thermodynamic aspects underlying the manipulated enantioselectivity of DR3 and DS6.

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Section: Discussionmentioning

confidence: 99%

Enhanced stereodivergent evolution of carboxylesterase for efficient kinetic resolution of near-symmetric esters through machine learning

Xu,

Dou,

Chen

et al. 2024

Preprint

Self Cite

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“…[19] In our recent study, structure-guided engineering of a BVMO from Rhodococcus aetherivorans (RaBVMO) was conducted for asymmetric sulfoxidation of OPS. [20] Whereas problems such as insufficient catalytic performance and unclear structure-function relationship still exist. Furthermore, except for AcCHMO and CHMO NCIMB9871 , no other CHMO has been discovered for the asymmetric synthesis of (S)-omeprazole, and that stereoselective inversion of CHMOs toward OPS has not yet been reported.…”

Section: Introductionmentioning

confidence: 99%

“…capable of producing ( S )‐omeprazole from OPS was obtained by substrate tunnel engineering [19] . In our recent study, structure‐guided engineering of a BVMO from Rhodococcus aetherivorans ( Ra BVMO) was conducted for asymmetric sulfoxidation of OPS [20] . Whereas problems such as insufficient catalytic performance and unclear structure‐function relationship still exist.…”

Section: Introductionmentioning

confidence: 99%

Structure‐Guided Evolution of Cyclohexanone Monooxygenase Toward Bulky Omeprazole Sulfide: Substrate Migration and Stereoselectivity Inversion

Wei,

Xu,

Zhou

et al. 2024

ChemPhysChem

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Structure‐guided engineering of a CHMO from Amycolatopsis methanolica (AmCHMO) was performed for asymmetric sulfoxidation activity and stereoselectivity toward omeprazole sulfide. Initially, combinatorial active‐site saturation test (CASTing) and iteratively saturation mutagenesis (ISM) were performed on 5 residues at the “bottleneck” of substrate tunnel, and MT3 was successfully obtained with a specific activity of 46.19 U/g and R‐stereoselectivity of 99% toward OPS. Then, 4 key mutations affecting the stereoselectivity were identified through multiple rounds of ISM on residues at the substrate binding pocket region, resulting MT8 with an inversed stereoselectivity from 99% (R) to 97% (S). MT8 has a greatly compromised specific activity of 0.08 U/g. By introducing additional beneficial mutations, MT11 was constructed with significantly increased specific activity of 2.29 U/g and stereoselectivity of 97% (S). Enlarged substrate tunnel is critical to the expanded substrate spectrum of AmCHMO, while reshaping of substrate binding pocket is important for stereoselective inversion.Based on MD simulation, pre‐reaction states of MT3‐OPSproR, MT8‐OPSproS, and MT11‐OPSproS were calculated to be 45.56%,17.94%, and 28.65% respectively, which further confirm the experimental data on activity and stereoselectivity. Our results pave the way for engineering distinct activity and stereoselectivity of BVMOs toward bulky prazole thioethers.

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Synthesis of Chiral Sulfoxides by A Cyclic Oxidation‐Reduction Multi‐Enzymatic Cascade Biocatalysis

Tian,

Zhou,

Chen

et al. 2024

Chemistry A European J

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Optically pure sulfoxides are valuable organosulfur compounds extensively employed in medicinal and organic synthesis. In this study, we present a biocatalytic oxidation‐reduction cascade system designed for the preparation of enantiopure sulfoxides. The system involves the cooperation of a low‐enantioselective chimeric oxidase SMO (styrene monooxygenase) with a high‐enantioselective reductase MsrA (methionine sulfoxide reductase A), facilitating “non‐selective oxidation and selective reduction” cycles for prochiral sulfide oxidation. The regeneration of requisite cofactors for MsrA and SMO was achieved via a cascade catalysis process involving three auxiliary enzymes, sustained by cost‐effective D‐glucose. Under the optimal reaction conditions, a series of heteroaryl alkyl, aryl alkyl and dialkyl sulfoxides in R configuration were synthesized through this “one‐pot, one step” cascade reaction. The obtained compounds exhibited high yields of >90% and demonstrated enantiomeric excess (ee) values exceeding 90%. This study represents an unconventional and efficient biocatalytic way in utilizing the low‐enantioselective oxidase for the synthesis of enantiopure sulfoxides.

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Engineering of a Baeyer–Villiger monooxygenase reveals key residues for the asymmetric oxidation of omeprazole sulfide

Abstract: Structure-guided engineering of a BVMO from Rhodococcus aetherivorans (RaBVMO) was performed for asymmetric sulfoxidation activity toward omeprazole sulfide. Based on the structrural model of RaBVMO, key residues lining the substrate...

Cited by 5 publications

References 32 publications

Enhanced stereodivergent evolution of carboxylesterase for efficient kinetic resolution of near-symmetric esters through machine learning

Enhanced stereodivergent evolution of carboxylesterase for efficient kinetic resolution of near-symmetric esters through machine learning

Structure‐Guided Evolution of Cyclohexanone Monooxygenase Toward Bulky Omeprazole Sulfide: Substrate Migration and Stereoselectivity Inversion

Synthesis of Chiral Sulfoxides by A Cyclic Oxidation‐Reduction Multi‐Enzymatic Cascade Biocatalysis

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