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Structural basis for halogenation by iron- and 2-oxo-glutarate-dependent enzyme WelO5

Nature Chemical Biology volume 12, pages 636–640 (2016)Cite this article

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Abstract

A 2.4-Å-resolution X-ray crystal structure of the carrier-protein-independent halogenase WelO5 in complex with its welwitindolinone precursor substrate, 12-epi-fischerindole U, reveals that the C13 chlorination target is proximal to the anticipated site of the oxo group in a presumptive cis-halo-oxo-iron(IV) (haloferryl) intermediate. Prior study of related halogenases forecasts substrate hydroxylation in this active-site configuration, but X-ray crystallographic verification of C13 halogenation in single crystals mandates that ligand dynamics must reposition the oxygen ligand to enable the observed outcome. S189A WelO5 produces a mixture of halogenation and hydroxylation products, showing that an outer-sphere hydrogen-bonding group orchestrates ligand movements to achieve a configuration that promotes halogen transfer.

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Figure 1: Structure of WelO5 in the absence of substrate.
Figure 2: The substrate-binding site in WelO5.
Figure 3: Functional characterization of the WelO5 substrate-binding site.
Figure 4: Proposed mechanism for WelO5-mediated halogenation and its structural basis.

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Acknowledgements

We thank R.J. Martinie, C. Krebs, and J.M. Bollinger, Jr. for critical discussions and reading of the manuscript. We acknowledge D. Smith, J. Brunzelle, and the staff at the CCP4/APS School for Macromolecular Crystallography for assistance with X-ray data collection and analysis. This work has been supported by US National Institutes of Health grant GM100011 (A.K.B.), the Searle Scholars Program (A.K.B.), and the Department of Chemistry at the University of Pittsburgh (X.L.). Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). GM/CA CAT has been funded in whole or in part with US federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1-GM-1104).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA

    Andrew J Mitchell, Ailiena O Maggiolo, Nikhil R Ananth & Amie K Boal

  2. Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Qin Zhu, Matthew L Hillwig & Xinyu Liu

  3. Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA

    Amie K Boal

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Contributions

A.J.M., A.O.M., and N.R.A. purified and crystallized protein samples and collected X-ray data sets. A.J.M. and A.O.M. processed X-ray data sets and solved structures. Q.Z. and M.L.H. prepared samples and performed biochemical experiments. A.J.M., X.L., and A.K.B. designed the experimental approach, analyzed data, and wrote the manuscript.

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Correspondence to Xinyu Liu or Amie K Boal.

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A.J.M., X.L., and A.K.B. have filed a patent application using information gained from this contribution.

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Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–20. (PDF 12955 kb)

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Mitchell, A., Zhu, Q., Maggiolo, A. et al. Structural basis for halogenation by iron- and 2-oxo-glutarate-dependent enzyme WelO5. Nat Chem Biol 12, 636–640 (2016). https://doi.org/10.1038/nchembio.2112

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