Reaction pathway engineering converts a radical hydroxylase into a halogenase

  • Nat Chem Biol. 2022 Feb;18(2):171-179. doi: 10.1038/s41589-021-00944-x.
Monica E Neugebauer  1 ,  Elijah N Kissman  2 ,  Jorge A Marchand  1 ,  Jeffrey G Pelton  3 ,  Nicholas A Sambold  4 ,  Douglas C Millar  1 ,  Michelle C Y Chang  5  6  7
Affiliations
  • 1. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA.
  • 2. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.
  • 3. QB3 Institute, University of California, Berkeley, Berkeley, CA, USA.
  • 4. Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
  • 5. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA. [email protected].
  • 6. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA. [email protected].
  • 7. Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA, USA. [email protected].
PMID: 34937913 DOI: 10.1038/s41589-021-00944-x
Abstract

FeII/α-ketoglutarate (FeII/αKG)-dependent Enzymes offer a promising biocatalytic platform for halogenation chemistry owing to their ability to functionalize unactivated C-H bonds. However, relatively few radical halogenases have been identified to date, limiting their synthetic utility. Here, we report a strategy to expand the palette of enzymatic halogenation by engineering a reaction pathway rather than substrate selectivity. This approach could allow us to tap the broader class of FeII/αKG-dependent hydroxylases as catalysts by their conversion to halogenases. Toward this goal, we discovered active halogenases from a DNA shuffle library generated from a halogenase-hydroxylase pair using a high-throughput in vivo fluorescent screen coupled to an alkyne-producing biosynthetic pathway. Insights from Sequencing halogenation-active variants along with the crystal structure of the hydroxylase enabled engineering of a hydroxylase to perform halogenation with comparable activity and higher selectivity than the wild-type halogenase, showcasing the potential of harnessing hydroxylases for biocatalytic halogenation.

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