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Structural and functional insights into asymmetric enzymatic dehydration of alkenols

Nature Chemical Biology volume 13, pages 275–281 (2017)Cite this article

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Abstract

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of β-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5–C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.

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Figure 1: Natural reaction and primary structure of LinD.
Figure 2: Structure of C. defragrans linalool dehydratase isomerase (LinD, PDB code 5G1W).
Figure 3: Mechanistic hypotheses for the dehydration and isomerization reaction catalyzed by LinD.

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Acknowledgements

This project has received funding from the European Union's Horizon 2020 research and innovation programme (EmPowerPutida) under grant agreement no. 635536 (to B.H.). This work was supported by the Diamond Light Source for access to beamlines I02 and I04 under proposed number mx-9948 (to G.G.).

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Authors and Affiliations

  1. Institute of Technical Biochemistry, Universitaet Stuttgart, Stuttgart, Germany

    Bettina M Nestl, Christopher Geinitz, Max-Philipp Fischer & Bernhard Hauer

  2. Department of Chemistry, York Structural Biology Laboratory, University of York, Heslington, York, UK

    Stephanie Popa, Hoi Ting Hau, Henry Man, Muhiadin Omar, Johan P Turkenburg & Gideon Grogan

  3. Genomatica, Inc., San Diego, California, USA

    Sari Rizek, Robert J Haselbeck, Rosary Stephen, Michael A Noble, Erik C Ralph, Stephen van Dien & Stephanie J Culler

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  1. Bettina M Nestl
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Contributions

S.P., H.T.H., H.M., M.O., J.P.T. and G.G. conceived and designed the crystallization experiments and performed 3D structure determination. S.R., R.J.H., R.S., M.A.N., E.C.R. S.V.D. and S.J.C. created and characterized all of the LinD mutants used. C.G., M.-P.F. and B.M.N. performed biotransformations and contributed to data analysis. B.M.N., G.G. and B.H. wrote the manuscript. All authors revised and contributed to the manuscript.

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Correspondence to Bernhard Hauer.

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Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–4. (PDF 828 kb)

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Synthetic Procedures. (PDF 640 kb)

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Nestl, B., Geinitz, C., Popa, S. et al. Structural and functional insights into asymmetric enzymatic dehydration of alkenols. Nat Chem Biol 13, 275–281 (2017). https://doi.org/10.1038/nchembio.2271

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