Journal
NATURE CHEMICAL BIOLOGY
Volume 18, Issue 2, Pages 171-+Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41589-021-00944-x
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Funding
- DOE/LBNL [DEAC02-05CH11231, FWP CH030201]
- National Science Foundation Graduate Research Fellowship
- National Institutes of Health NRSA Training Grant [1 T32 GMO66698]
- UC Berkeley Chancellor's Fellowship
- Howard Hughes Medical Institute Gilliam Fellowship
- University of California Office of the President, Multicampus Research Programs and Initiatives [MR-15- 328599]
- National Institutes of Health [R01 GM124149, P30 GM124169]
- Plexxikon
- Integrated Diffraction Analysis Technologies program of the US Department of Energy Office of Biological and Environmental Research
- US Department of Energy, Office of Basic Energy Sciences [DEAC02-05CH11231]
- NIH [GM68933]
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Researchers have developed a strategy to expand enzymatic halogenation by engineering a reaction pathway rather than substrate selectivity. They discovered active halogenases from a DNA shuffle library and engineered 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.
Fe-II/alpha-ketoglutarate (Fe-II/alpha KG)-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 Fe-II/alpha KG-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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