Identification of key amino acid residues determining product specificity of 2,3‐oxidosqualene cyclase in Oryza species

Abstract

Triterpene synthases, also known as 2,3-oxidosqualene cyclases (OSCs), synthesize diverse triterpene skeletons that form the basis of an array of functionally divergent steroids and triterpenoids. Tetracyclic and pentacyclic triterpene skeletons are synthesized via protosteryl and dammarenyl cations, respectively. The mechanism of conversion between two scaffolds is not well understood. Here, we report a promiscuous OSC from rice (Oryza sativa) (OsOS) that synthesizes a novel pentacyclic triterpene orysatinol as its main product. The OsOS gene is widely distributed in indica subspecies of cultivated rice and in wild rice accessions. Previously, we have characterized a different OSC, OsPS, a tetracyclic parkeol synthase found in japonica subspecies. Phylogenetic and protein structural analyses identified three key amino acid residues (#732, #365, #124) amongst 46 polymorphic sites that determine functional conversion between OsPS and OsOS, specifically, the chair-semi(chair)-chair and chair-boat-chair interconversions. The different orientation of a fourth amino acid residue Y257 was shown to be important for functional conversion The discovery of orysatinol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into tetra- and pentacyclic skeletons, and provide a new strategy to identify key residues determining OSC specificity.

Oryza sativa Parkeol Cyclase: Changes in the Substrate‐Folding Conformation and the Deprotonation Sites on Mutation at Tyr257: Importance of the Hydroxy Group and Steric Bulk

“…They constructed three site‐directed mutants—Y257L, Y257F, and Y257A—and reported that only trace amounts of parkeol were produced through the action of these mutants. No other products were reported . The results reported by them are significantly different from those of our experiments; these differences might be due to a lack of detailed experiments by them.…”

“…Xue et al. recently reported mutagenesis experiments targeting the Tyr257 residue of parkeol synthase ( Os PA, Os OSC2) . They constructed three site‐directed mutants—Y257L, Y257F, and Y257A—and reported that only trace amounts of parkeol were produced through the action of these mutants.…”

The protosteryl and dammarenyl cation dichotomy in polycyclic triterpene biosynthesis revisited: has this ‘rule’ finally been broken?

“…4a). This is supported by site-directed mutagenesis work 52 which report the functional conversion of a committed parkeol synthase to a variant producing orysatinol (48). Further support comes from phylogenetic analysis suggesting that the wild type orysatinol synthase shares a common (parkeol (8) producing) ancestor with the committed parkeol synthase.…”

Friedelane‐type triterpene cyclase in celastrol biosynthesis from Tripterygium wilfordii and its application for triterpenes biosynthesis in yeast

“…The mechanistic diversity of OSCs remains intriguing. Although many attempts have been made to study the underlying mechanism (Hart et al ., ; Herrera et al ., ; Tetsuo Kushiro et al ., ; Liu et al ., ; Salmon et al ., ; Xue et al ., ), the mechanism of conversion between scaffolds remains poorly understood. Sequence analysis has shown that all three Tw OSCs contained terpenoid cyclase/protein prenyltransferase alpha–alpha toroid domains, but only Tw OSC2 contained conserved sites of terpene synthase that are rich in aromatic residues, in the C‐terminal region.…”