Reaction pathway engineering converts a radical hydroxylase into a halogenase.
Nat Chem Biol
; 18(2): 171-179, 2022 02.
Article
de En
| MEDLINE
| ID: mdl-34937913
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.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Halogènes
/
Mixed function oxygenases
Langue:
En
Journal:
Nat Chem Biol
Sujet du journal:
BIOLOGIA
/
QUIMICA
Année:
2022
Type de document:
Article
Pays d'affiliation:
États-Unis d'Amérique