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

Nature Catalysis volume 5, pages 534–544 (2022)Cite this article

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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.

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Fig. 1: Mechanism-guided engineering approach to increase efficiency of a FDH.
Fig. 2: A tunnel connecting the two active sites of Thal and engineering results.
Fig. 3: Enzymatic properties of Thal-V82I compared with Thal-WT.
Fig. 4: Molecular dynamics simulations explain the decrease of HOBr leakage in Thal-V82I.
Fig. 5: Transient kinetics analysis of individual steps of Thal-WT and Thal-V82I.
Fig. 6: A wide substrate scope for Thal-V82I.

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Data availability

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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Acknowledgements

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.

Author information

Author notes

  1. These authors contributed equally: Kridsadakorn Prakinee, Aisaraphon Phintha.

Authors and Affiliations

  1. School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Wangchan Valley, Rayong, Thailand

    Kridsadakorn Prakinee, Aisaraphon Phintha, Surawit Visitsatthawong, Chadaporn Kantiwiriyawanitch & Pimchai Chaiyen

  2. Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, Thailand

    Aisaraphon Phintha

  3. Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand

    Narin Lawan

  4. Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand

    Jeerus Sucharitakul

  5. Loschmidt Laboratories, RECETOX, Faculty of Science and Loschmidt Laboratories, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

    Jiri Damborsky

  6. International Clinical Research Center, St. Anne’s University Hospital Brno, Brno, Czech Republic

    Jiri Damborsky

  7. Biomolecular Analysis and Application Research Team, National Center for Genetic Engineering and Biotechnology, Klong Luang, Pathumthani, Thailand

    Penchit Chitnumsub

  8. General Biochemistry, Technical University of Dresden, Dresden, Germany

    Karl-Heinz van Pée

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Contributions

K.P., A.P. and P. Chaiyen conceived and designed the study. A.P., S.V. and K.P. performed the tunnel analysis and rational design of enzyme engineering. K.P. and A.P. conducted the library creation and screening. K.P., A.P. and C.K. performed protein production and purification, and enzymatic assays. K.P. and A.P. performed the transient kinetics experiments with contributions from J.S. N.L. performed the computational analysis. S.V., K.P. and N.L. analysed the molecular dynamics simulations. S.V., N.L., J.S., J.D., P. Chitnumsub, and K.-H.v.P., analysed data and reviewed the manuscript. K.P., A.P. and P. Chaiyen prepared the manuscript.

Corresponding author

Correspondence to Pimchai Chaiyen.

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Nature Catalysis thanks Roland Ludwig, Christian Schnepel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Methods, Table 1, Figs. 1–57 and References.

Reporting Summary

Supplementary Data 1

Initial structure molecular dynamics of WT.

Supplementary Data 2

Molecular dynamics snapshot of WT_400K at 4 ns.

Supplementary Data 3

Initial structure molecular dynamics of V82I.

Supplementary Data 4

Molecular dynamics snapshot of V82I_400K at 4 ns.

Supplementary Data 5

Initial structure molecular dynamics of WT_HOBr.

Supplementary Data 6

Molecular dynamics snapshot of WT_HOBr at 19.4 ns.

Supplementary Data 7

Initial structure molecular dynamics of V82I_HOBr.

Supplementary Data 8

Molecular dynamics snapshot of V82I_HOBr at 19.4 ns.

Supplementary Data 9

Molecular dynamics snapshot of V82I_HOBr at 102 ns.

Supplementary Data 10

Initial structure molecular dynamics of WT_Phenol.

Supplementary Data 11

Molecular dynamics snapshot of WT_Phenol at 8 ns.

Supplementary Data 12

Initial structure molecular dynamics of V82I_Phenol.

Supplementary Data 13

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

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