Cytochrome P450 Mediated Cyclohexane Ring Formation in Forazoline Biosynthesis.

Area of Science:

• Natural Product Biosynthesis
• Enzymology
• Structural Biology

Background:

• Forazoline A is a unique PK/NRP hybrid macrolactone with potent in vivo antifungal activity.
• The precise biosynthetic pathway and tailoring enzyme functions for Forazoline A remain largely uncharacterized.
• A PKS/NRPS gene cluster was previously identified as a candidate for Forazoline A production.

Purpose of the Study:

• To characterize the functions of three cytochrome P450 mono-oxygenases (FrazP1P2P3) involved in Forazoline A biosynthesis.
• To elucidate the mechanism of FrazP2 in catalyzing a key cyclization step.
• To gain structural and mechanistic insights into FrazP2 activity.

Main Methods:

• Genetic analysis and biochemical assays to confirm FrazP2 function.
• X-ray crystallography to determine the structure of a FrazP2-substrate complex.
• Molecular dynamics simulations and DFT calculations to analyze the reaction mechanism.

Main Results:

• FrazP2 was identified as the enzyme responsible for catalyzing cyclohexane ring formation from an 1,3,6-triene precursor.
• The crystal structure of FrazP2 in complex with its substrate was solved at 2.3 Å resolution.
• Enzyme simulations revealed an unprecedented oxidative cyclization reaction catalyzed by FrazP2.

Conclusions:

• The characterization of FrazP2 expands the understanding of cytochrome P450 catalytic diversity.
• These findings contribute to the diversification of natural products and the potential creation of novel antifungal agents.
• The study provides a foundation for engineering unnatural derivatives of Forazoline A with enhanced antifungal potency.