Enzymatic Redox Gating Directs Oxidative Divergence in Acyclic Peroxides Biosynthesis.

Area of Science:

• Biochemistry
• Natural Product Biosynthesis
• Enzymology

Background:

• Peroxy natural products are vital compounds with diverse biological roles.
• The enzymatic pathways for synthesizing O─O bonds in peroxides are largely unknown.
• Few enzymes forming endoperoxides have been identified, and none for acyclic peroxides.

Purpose of the Study:

• To elucidate the enzymatic mechanisms behind acyclic peroxide formation in natural product biosynthesis.
• To identify novel enzymes responsible for catalyzing peroxide installation.
• To understand the regulatory strategies controlling oxidative enzymatic reactions.

Main Methods:

• Identification and characterization of two homologous flavin-dependent enzymes, OxaJ and OtnJ.
• Biochemical assays to determine enzyme activity and substrate specificity.
• Structural analysis to understand the mechanism of peroxide formation and redox gating.

Main Results:

• OxaJ and OtnJ were identified as flavin-dependent enzymes catalyzing enantioselective acyclic peroxide formation in oxanthromicin biosynthesis.
• A conserved structural motif was found to function as a redox gate, controlling NADPH access to the active site.
• Removal of the redox gate motif altered enzyme activity, redirecting it towards hydroxylation instead of peroxide formation.

Conclusions:

• This study presents the first example of peroxide formation catalyzed by a flavin-dependent enzyme.
• Redox gating is identified as a novel mechanism for controlling oxidative divergence in enzymatic catalysis.
• The findings provide critical insights into the biosynthesis of peroxy natural products and enzyme regulatory strategies.