Dissection of the Catalytic Mechanisms of Transmembrane Terpene Cyclases Involved in Fungal Meroterpenoid Biosynthesis

Abstract Pyr4‐family terpene cyclases are noncanonical transmembrane terpene cyclases involved in the biosynthesis of microbial meroterpenoids and catalyze diverse cyclization reactions. Despite the ubiquity of Pyr4‐family terpene cyclases in microorganisms, their three‐dimensional structures have never been experimentally determined. Herein, we focused on AdrI, the Pyr4‐family enzyme for the andrastin A pathway, and its homologues, and performed a series of mutational experiments using their AlphaFold2‐generated structures. Intriguingly, we found that AdrI and InsA7, which both accept the same substrate, use different amino acid residues for the initiation of the cyclization cascade. Furthermore, we obtained several AdrI variants with altered product selectivity, one of which dominantly yielded a new meroterpenoid species. Collectively, our study provides important insights into the catalytic functions of Pyr4‐family terpene cyclases and will facilitate the engineering of these enzymes.