A two-metalloenzyme cascade constructs the azetidine-containing pharmacophore

Area of Science:

• Biochemistry
• Enzymology
• Organic Chemistry

Background:

• Azetidine is a crucial pharmacophore found in numerous natural and synthetic drug molecules.
• The biosynthetic pathway for azetidine formation in nature has remained largely unknown.

Purpose of the Study:

• To elucidate the complete enzymatic cascade responsible for synthesizing polyoximic acid, a molecule containing the azetidine moiety.
• To characterize the roles and mechanisms of the metalloenzymes involved in this pathway.

Main Methods:

• Biochemical characterization of the PolE and PolF enzymes.
• Enzyme assays involving L-isoleucine as a substrate.
• Structural studies using X-ray crystallography.
• Quantum mechanics/molecular mechanics (QM/MM) simulations.

Main Results:

• PolE was identified as an Fe(II)/pterin-dependent L-isoleucine desaturase.
• PolF was characterized as a novel haem-oxygenase-like diiron oxidase.
• PolF catalyzes the conversion of desaturated L-isoleucine to polyoximic acid via intramolecular C-N cyclization.
• PolF exhibits dual functionality, performing both desaturation and cyclization sequentially.
• Structural and QM/MM studies revealed an extraordinary mechanism for azetidine ring formation by PolF.

Conclusions:

• The study deciphers a two-metalloenzyme cascade for azetidine biosynthesis, specifically for polyoximic acid.
• The findings reveal novel functions for metalloenzymes, including the dual role of PolF.
• This work provides insights into metalloenzyme catalysis and offers a basis for the rational design of azetidine-containing molecules.