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A family of radical halogenases for the engineering of amino-acid-based products

Nature Chemical Biology volume 15, pages 1009–1016 (2019)Cite this article

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Abstract

The integration of synthetic and biological catalysis enables new approaches to the synthesis of small molecules by combining the high selectivity of enzymes with the reaction diversity offered by synthetic chemistry. While organohalogens are valued for their bioactivity and utility as synthetic building blocks, only a handful of enzymes that carry out the regioselective halogenation of unactivated \({\rm{C}}_{sp^3}{-}{\rm{H}}\) bonds have previously been identified. In this context, we report the structural characterization of BesD, a recently discovered radical halogenase from the FeII/α-ketogluturate-dependent family that chlorinates the free amino acid lysine. We also identify and characterize additional halogenases that produce mono- and dichlorinated, as well as brominated and azidated, amino acids. The substrate selectivity of this new family of radical halogenases takes advantage of the central role of amino acids in metabolism and enables engineering of biosynthetic pathways to afford a wide variety of compound classes, including heterocycles, diamines, α-keto acids and peptides.

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Fig. 1: Crystal structure of lysine halogenase BesD.
Fig. 2: Proposed mechanism of halogenation by BesD.
Fig. 3: Alanine scan of active site residues.
Fig. 4: Amino acid halogenase diversity.
Fig. 5: Engineering downstream pathways with amino acid halogenases.

Data availability

Accession codes for proteins in this study are provided in Supplementary Table 2. The PDB accession code for the BesD structure is 6NIE. Source data are available online for Figs. 3–5. Datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This work received support from the National Science Foundation (CHE-1710588) and the Department of Energy (DOE/LBNL DEAC02-05CH11231, FWP CH030201). M.E.N. acknowledges the support of a National Science Foundation graduate research fellowship. J.L.M. acknowledges the support of a National Institutes of Health NRSA training grant (1 T32 GMO66698). J.A.M. acknowledges the support of a University of California, Berkeley Chancellor’s fellowship, Howard Hughes Medical Institute Gilliam fellowship and National Institutes of Health NRSA training grant (1 T32 GMO66698). X-ray data were collected at the Advanced Light Source Beamline 8.3.1, which is operated by the University of California Office of the President, Multicampus Research Programs and Initiatives (MR-15-328599), the National Institutes of Health (R01 GM124149 and P30 GM124169), Plexxikon and the Integrated Diffraction Analysis Technologies program of the U.S. Department of Energy Office of Biological and Environmental Research. The Advanced Light Source is a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the U.S. Department of Energy under contract number DEAC02-05CH11231, Office of Basic Energy Sciences. The funds for the 900-MHz NMR spectrometer housed in the QB3 Institute in Stanley Hall at University of California, Berkeley were provided by the National Institutes of Health (GM68933). We thank E. C. Wittenborn, J. Holton, C. Gee and G. Meigs for crystallography advice. We also thank J.M. Bollinger and A.K. Boal for helpful discussions.

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA

    Monica E. Neugebauer, Jorge A. Marchand & Michelle C. Y. Chang

  2. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    Kiera H. Sumida, Jonathan L. McMurry & Michelle C. Y. Chang

  3. QB3 Institute, University of California, Berkeley, Berkeley, CA, USA

    Jeffrey G. Pelton

  4. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Michelle C. Y. Chang

  5. Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Michelle C. Y. Chang

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  1. Monica E. Neugebauer
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  2. Kiera H. Sumida
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  4. Jonathan L. McMurry
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Contributions

M.E.N. carried out protein crystallography, bioinformatics and enzyme characterization experiments. K.H.S. carried out enzyme characterization experiments. J.G.P. performed NMR experiments. J.L.M. contributed to bioinformatics. J.A.M. contributed helpful discussions and contributed to bioinformatics. M.E.N., M.C.Y.C. and K.H.S. planned experiments. M.E.N. and M.C.Y.C. wrote the manuscript.

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Correspondence to Michelle C. Y. Chang.

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Supplementary Tables 1–5, Supplementary Figures 1–23 and Supplementary Note 2

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Neugebauer, M.E., Sumida, K.H., Pelton, J.G. et al. A family of radical halogenases for the engineering of amino-acid-based products. Nat Chem Biol 15, 1009–1016 (2019). https://doi.org/10.1038/s41589-019-0355-x

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