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Chem Catalysis
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Analysis of laboratory-evolved flavin-dependent halogenases affords a computational model for predicting halogenase site selectivity

  • Author Footnotes
    5 Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
    ,
    Author Footnotes
    8 These authors contributed equally
    Mary C. Andorfer
    Footnotes
    5 Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
    8 These authors contributed equally
    Affiliations
    Department of Chemistry, University of Chicago, Chicago, IL 60637, USA
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  • Author Footnotes
    8 These authors contributed equally
    Declan Evans
    Footnotes
    8 These authors contributed equally
    Affiliations
    Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA
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  • Author Footnotes
    6 Present address: Merck Research Laboratories, South San Francisco, CA, USA
    Song Yang
    Footnotes
    6 Present address: Merck Research Laboratories, South San Francisco, CA, USA
    Affiliations
    Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA
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  • Author Footnotes
    7 Present address: Merck Research Laboratories, Kenilworth, NJ, USA
    Cyndi Qixin He
    Footnotes
    7 Present address: Merck Research Laboratories, Kenilworth, NJ, USA
    Affiliations
    Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA
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  • Anna M. Girlich
    Affiliations
    Department of Chemistry, University of Chicago, Chicago, IL 60637, USA
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  • Jaylie Vergara-Coll
    Affiliations
    Department of Chemistry, University of Chicago, Chicago, IL 60637, USA
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  • Narayanasami Sukumar
    Affiliations
    NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Building 436E, Argonne, IL 60439, USA
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  • K.N. Houk
    Correspondence
    Corresponding author
    Affiliations
    Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA
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  • Author Footnotes
    9 Lead contact
    Jared C. Lewis
    Correspondence
    Corresponding author
    Footnotes
    9 Lead contact
    Affiliations
    Department of Chemistry, Indiana University, Bloomington, IN 47405, USA
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  • Author Footnotes
    5 Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
    6 Present address: Merck Research Laboratories, South San Francisco, CA, USA
    7 Present address: Merck Research Laboratories, Kenilworth, NJ, USA
    8 These authors contributed equally
    9 Lead contact

      Highlights

      • Flavin-dependent halogenases site-selectively halogenate under mild conditions
      • Selectivity of evolved variants explored using crystallography and mutagenesis
      • Data are consistent with hypohalous acid as the active halogenating species
      • Predictive computational model for halogenase site selectivity

      Summary

      Flavin-dependent halogenases (FDHs) catalyze selective halogenation of electron-rich aromatic compounds without the need for harsh oxidants required by conventional oxidative halogenation reactions. Predictive models for halogenase site selectivity could greatly improve their utility for chemical synthesis. Toward this end, we analyzed the structures and selectivity of three halogenase variants evolved to halogenate tryptamine with orthogonal selectivity. Crystal structures and reversion mutations revealed key residues involved in altering halogenase selectivity. Density functional theory calculations and molecular dynamics simulations are both consistent with hypohalous acid as the active halogenating species in FDH catalysis. This model was used to accurately predict the site selectivity of halogenase variants toward different synthetic substrates, providing a valuable tool for implementing halogenases in biocatalysis efforts.

      Graphical abstract

      Keywords

      UN Sustainable Development Goals

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