Mechanism-Guided Tunnel Engineering to Increase the Efficiency of a Flavin-Dependent Halogenase

Authors

Prakinee, K., Phintha, A., Visitsatthawong, S., Lawan, N., Sucharitakul, J., Kantiwiriyawanitch, C., Damborsky, J., Chitnumsub, P., van Pée, K.-H., Chaiyen, P.

Source

NATURE CATALYSIS 5: 534-544 (2022)

Abstract

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.

Full text •  Supplement

Citation

Prakinee, K., Phintha, A., Visitsatthawong, S., Lawan, N., Sucharitakul, J., Kantiwiriyawanitch, C., Damborsky, J., Chitnumsub, P., van Pée, K.-H., Chaiyen, P., 2022: Mechanism-Guided Tunnel Engineering to Increase the Efficiency of a Flavin-Dependent Halogenase. Nature Catalysis 5: 534-544.

Lukas’s Success at SOC Competition
3rd Hands-on Computational Enzyme Design Course Successfully Behind Us
Veronika and Honza to Present at Cerebrovascular Congress
Success at RECETOX PhD Conference
Daniel’s Success at Interdisciplinary Meeting in Milovy
Lukas Awarded at SOC
Dean’s Award for Daniel, Michal and Martin