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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The initial structures and snapshots of molecular dynamics simulations are given as Supplementary Data and available at https://github.com/N-Lawan/Flavin-dependent-halogenase.git. The data supporting the findings of this study are available within the article and its Supplementary Information or can be obtained from the corresponding author on reasonable request.
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We thank the Thailand Science Research Innovation and NSRF via the Program Management Unit for Human Resources and Institutional Development, Research and Innovation (grant no. B05F640089) and the Royal Academy of Engineering (for their support to P. Chaiyen), the Vidyasirimedhi Institute of Science and Technology (VISTEC) (for their support to K.P., A.P., S.V., C.K. and P. Chaiyen), the Thailand Science Research Innovation and National Research Council of Thailand (Royal Golden Jubilee PHD/0135/2557 grant to A. P. and P. Chaiyen) and Chiang Mai University for partial support to N. Lawan. We acknowledge the VISTEC-NSTDA fellowship (to C. Kantiwiriyawanitch, P. Chaiyen and P. Chitnumsub), and thank the Czech Ministry of Education for financial support to J. Damborsky (grant nos. CZ.02.1.01/0.0/0.0/16_026/0008451 and LM2018121). We thank S. Maenpuen (Burapha University) for providing stopped-flow and rapid-quench flow technical support, and V. Pongsupasa (VISTEC) for providing a thermostable C1-A58P enzyme for the thermostability assays. We thank U. Bornscheuer and M. Dörr (University of Greifswald) for valuable advice related to enzyme engineering procedures. We thank S. Ketrat, S. Nutanong and School of Information Science and Technology, VISTEC for computing facilities. The figures were created using materials from PyMOL and BioRender.com.
The authors declare no competing interests.
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Supplementary Methods, Table 1, Figs. 1–57 and References.
Initial structure molecular dynamics of WT.
Molecular dynamics snapshot of WT_400K at 4 ns.
Initial structure molecular dynamics of V82I.
Molecular dynamics snapshot of V82I_400K at 4 ns.
Initial structure molecular dynamics of WT_HOBr.
Molecular dynamics snapshot of WT_HOBr at 19.4 ns.
Initial structure molecular dynamics of V82I_HOBr.
Molecular dynamics snapshot of V82I_HOBr at 19.4 ns.
Molecular dynamics snapshot of V82I_HOBr at 102 ns.
Initial structure molecular dynamics of WT_Phenol.
Molecular dynamics snapshot of WT_Phenol at 8 ns.
Initial structure molecular dynamics of V82I_Phenol.
Molecular dynamics snapshot of V82I_Phenol at 8 ns.
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Prakinee, K., Phintha, A., Visitsatthawong, S. et al. Mechanism-guided tunnel engineering to increase the efficiency of a flavin-dependent halogenase. Nat Catal 5, 534–544 (2022). https://doi.org/10.1038/s41929-022-00800-8
Biophysical Reviews (2022)