Premutilin Synthase: Ring Rearrangement by a Class II Diterpene Cyclase

Area of Science:

• Biochemistry
• Organic Chemistry
• Molecular Biology

Background:

• Pleuromutilin is a complex diterpenoid antibiotic.
• Its biosynthesis involves a bifunctional (di)terpene synthase with two distinct active sites.

Purpose of the Study:

• To elucidate the role of each active site in pleuromutilin biosynthesis.
• To characterize the intermediate products formed during the reaction.

Main Methods:

• Site-directed mutagenesis was employed to selectively disable each active site of the bifunctional enzyme.
• The resulting intermediates were analyzed to understand the reaction mechanism.
• Quantum chemical calculations were performed to investigate the reaction's stereochemical requirements.

Main Results:

• Disabling the class I terpene synthase active site allowed for the characterization of the intermediate produced by the class II diterpene cyclase active site.
• A novel ring-contracted intermediate was identified, formed by the initiating class II cyclase.
• Quantum chemical calculations supported the significance of reactant configuration in the observed ring rearrangement.

Conclusions:

• The study successfully dissected the functions of the two active sites within the bifunctional terpene synthase.
• A previously unknown ring-contracted intermediate in pleuromutilin biosynthesis was discovered.
• The findings emphasize the critical role of reactant configuration in driving the complex diterpenoid rearrangement.