Molecular Basis for Sesterterpene Diversity Produced by Plant Terpene Synthases

Abstract

Class I terpene synthase (TPS) generates bioactive terpenoids with diverse backbones. Sesterterpene synthase (sester-TPS, C25), a branch of class I TPSs, was recently identified in Brassicaceae. However, the catalytic mechanisms of sester-TPSs are not fully understood. Here, we first identified three nonclustered functional sester-TPSs (AtTPS06, AtTPS22, and AtTPS29) in Arabidopsis thaliana . AtTPS06 utilizes a type-B cyclization mechanism, whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism. We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs. We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism: (1) mainly due to the outward shift of helix G, plant sester-TPSs have a larger catalytic pocket than do mono-, sesqui-, and di-TPSs to accommodate GFPP; (2) type-A sester-TPSs have more aromatic residues (five or six) in their catalytic pocket than classic TPSs (two or three), which also determines whether the type-A or type-B cyclization mechanism is active; and (3) the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs. Altogether, this study improves our understanding of the catalytic mechanism of plant sester-TPS, which ultimately enables the rational engineering of sesterterpenoids for future applications.

Occurrence and biosynthesis of plant sesterterpenes (C25), a new addition to terpene diversity

“…Phylogenetic analysis of plant TPS sequences clearly shows that functional sesterTPSs evolved from the TPS-a subfamily, the members of which always utilize GPP and/or FPP as substrates ( Chen et al., 2019 ). This result is consistent with the expansion of the TPS-a subfamily in Brassicaceae, as there are 22 TPS-a members (32 TPSs in total) in Arabidopsis ( Chen et al., 2011 , 2020 ). Because plant sesterTPSs have been identified only from Brassicaceae, it remains unclear whether the sesterTPSs from other plant species (e.g., Lamiaceae) evolved from the TPS-a subfamily or from other TPS subfamilies.…”

“…(2019) (−)-arathanadiene A ( C5 ) (−)-arathanadiene B ND AtTPS17 Huang et al. (2018) ( 2E )-flocerene ND AtTPS06 Chen et al. (2020) Capsella rubella (3, Brassicaceae) (−)-caprutriene A and C (−)-caprudiene A ND Cr237 , Cr089 (sesterTPS) Huang et al.…”

“…With the rapid development of genome mining and biochemical methods in Arabidopsis and other Brassicaceae plants ( Brassica rapa , Brassica napus , Brassica oleracea , Camelina sativa , and Capsella grandiflora ), approximately 20 sesterterpene skeletons have been produced and identified by reconstruction of their biosynthetic pathway in either Escherichia coli or tobacco ( Nicotiana benthamiana ) ( Huang et al., 2017 , 2018 ; Shao et al., 2017 ; Chen et al., 2019 , 2020 ). Notably, only four sesterterpene skeletons were detected ( C6–C9 , Figure 1 ) in Arabidopsis , and to the best of our knowledge, no oxidized sesterterpenes have been identified in Brassicaceae plants to date ( Huang et al., 2017 ; Chen et al., 2019 ).…”

“…Due to the presence of GFPPS - TPS - P450 clusters in the Arabidopsis genome ( Figure 2 B), it is reasonable to investigate whether the TPSs located in these biosynthetic gene clusters (BGCs) utilize GFPP as a substrate to produce sesterterpene backbones, even though no sesterterpene has been reported previously in Arabidopsis . Using different expression systems, 18 Brassicaceae - specific sesterTPSs were recently characterized, and 20 sesterterpene products were purified and elucidated ( Huang et al., 2017 , 2018 ; Shao et al., 2017 ; Chen et al., 2019 , 2020 ). The main products of AtTPS18 and AtTPS19 were first identified among these sesterterpenes ( Shao et al., 2017 ).…”

Systematic mining of fungal chimeric terpene synthases using an efficient precursor-providing yeast chassis

“…They are produced by typical plant TSs, which are only distantly related to microbial TSs ( 33 ). The plant sesterterpene synthases are newly derived members within the TPS-a subfamily, the general function of which is sesterterpene biosynthesis ( 48 ). This presents a case of neofunctionalization.…”