Mechanism-guided tunnel engineering to increase the efficiency of a flavin-dependent halogenase

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Authors

PRAKINEE Kridsadakorn PHINTHA Aisaraphon VISITSATTHAWONG Surawit LAWAN Narin SUCHARITAKUL Jeerus KANTIWIRIYAWANITCH Chadaporn DAMBORSKÝ Jiří CHITNUMSUB Penchit KARL-HEINZ Van Pee CHAIYEN Pimchai

Year of publication 2022
Type Article in Periodical
Magazine / Source NATURE CATALYSIS
MU Faculty or unit

Faculty of Science

Citation
Web https://www.nature.com/articles/s41929-022-00800-8
Doi http://dx.doi.org/10.1038/s41929-022-00800-8
Keywords TRYPTOPHAN 7-HALOGENASE; BIOCATALYTIC SCOPE; MOLECULAR-DYNAMICS; KINETIC MECHANISM; CHLORINATION; BIOSYNTHESIS; INTERMEDIATE; INSIGHTS; PHENOL; REBH
Description Although flavin-dependent halogenases (FDHs) are attractive for C-H bond activation, their applications are limited due to low turnover and stability. We have previously shown that leakage of a halogenating intermediate, hypohalous acid (HOX), causes FDHs to be inefficient by lessening halogenation yield. Here we employed a mechanism-guided semi-rational approach to engineer the intermediate transfer tunnel connecting two active sites of tryptophan 6-halogenase (Thal). This Thal-V82I variant generates less HOX leakage and possesses multiple catalytic improvements such as faster halogenation, broader substrate utilization, and greater thermostability and pH tolerance compared with the wildtype Thal. Stopped-flow and rapid quench kinetics analyses indicated that rate constants of halogenation and flavin oxidation are faster for Thal-V82I. Molecular dynamics simulations revealed that the V82I substitution introduces hydrophobic interactions which regulate tunnel dynamics to accommodate HOX and cause rearrangement of water networks, allowing better use of various substrates than the wildtype. Our approach demonstrates that an in-depth understanding of reaction mechanisms is valuable for improving efficiency of FDHs.
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