Angewandte Chemie International Edition
10.1002/anie.201706342
COMMUNICATION
to phenol hydroxyl group. Two active site residues, K74 and
D325, were shown to be essential for RadH activity and from
this a mechanism is proposed (Figure 3) that is supported by the
observation that compounds possessing methoxy groups, in
place of the substrate phenolic hydroxyl, are not halogenated by
RadH. In addition, we have developed a high throughput
fluorescence assay, which was used to screen for a RadH
mutant that exhibited higher activity. Deployment of the
improved RadH variant into a coumarin biosynthetic pathway
assembled in an E. coli host strain, demonstrates that
biosynthetic enzymes from diverse plant, bacterial and fungal
origins can be combined to create new pathways to novel
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Figure 4. Engineered pathway to 8-chloro-7-hydroxycoumarin 8a in E. coli.
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Keywords: Halogenase • Enzyme Mechanism • Directed
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