2. Academic Validation
  3. Reaction pathway engineering converts a radical hydroxylase into a halogenase

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

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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