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Angew Chem Int Ed Engl. 2017 Sep 18;56(39):11841-11845. doi: 10.1002/anie.201706342. Epub 2017 Aug 18.

RadH: A Versatile Halogenase for Integration into Synthetic Pathways.

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1
School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.

Abstract

Flavin-dependent halogenases are useful enzymes for providing halogenated molecules with improved biological activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site-directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high-throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin "non-natural" product in E. coli.

KEYWORDS:

biocatalysis; directed evolution; enzyme mechanisms; halogenases; pathway engineering

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