Non-Canonical Type II Terpene Cyclases Deliver Rare Terpenoid Architectures

Area of Science:

• Biochemistry
• Natural Product Biosynthesis
• Enzymology

Background:

• Terpene cyclases (TCs) are key enzymes in synthesizing complex terpenoids.
• Type II TCs and their role in indole diterpenoid (IDT) biosynthesis are poorly understood.
• Radarins are insecticidal IDTs with a unique structure, and their biosynthetic pathway was unknown.

Purpose of the Study:

• To elucidate the biosynthetic machinery responsible for the radarin scaffold in *Aspergillus fresenii*.
• To characterize the function of a novel IDT epoxidase (RadM) and a noncanonical type II TC (RadB).
• To understand the mechanism of complex skeletal rearrangement in IDT formation.

Main Methods:

• Genomic interrogation of *Aspergillus fresenii* to identify biosynthetic genes.
• Coexpression of identified genes (RadM and RadB) to reconstitute IDT biosynthesis.
• Biochemical assays and molecular modeling to study enzyme function and mechanism.

Main Results:

• Identified RadM, an epoxidase acting on 3'-geranylgeranylindole to produce a specific IDT precursor.
• Demonstrated coexpression of RadM and RadB yields radarin C through a complex rearrangement involving alcoholic deprotonation and ketone formation.
• Revealed a unique catalytic His residue in RadB essential for its function.
• Coexpression with an alternative TC produced a novel decalin IDT from an epoxide precursor.

Conclusions:

• Solved the biosynthetic puzzle for radarin scaffold formation, a key class of IDTs.
• Provided insights into the engineering of type II terpene cyclases.
• Expanded the known chemical diversity and catalytic capabilities of noncanonical terpene cyclases.