title: Molecular evolution of the substrate specificity of ent-kaurene synthases to adapt to gibberellin biosynthesis in land plants.
abstract: ent-Kaurene is a key intermediate in the biosynthesis of the plant hormone gibberellin. In ent-kaurene biosynthesis in flowering plants, two diterpene cyclases (DTCs), ent-copalyl diphosphate (ent-CDP) synthase (ent-CPS) and ent-kaurene synthase (KS), catalyse the cyclization of geranylgeranyl diphosphate to ent-CDP and ent-CDP to ent-kaurene, respectively. In contrast, the moss Physcomitrella patens has a bifunctional ent-CPS/KS (PpCPS/KS) that catalyses both cyclization reactions. To gain more insight into the functional diversity of ent-kaurene biosynthetic enzymes in land plants, we focused on DTCs in the lycophyte Selaginella moellendorffii. The present paper describes the characterization of two S. moellendorffii DTCs (SmKS and SmDTC3) in vitro. SmDTC3 converted ent-CDP into ent-16α-hydroxykaurane and also used other CDP stereoisomers as substrate. Remarkably, SmKS, which produces ent-kaurene from ent-CDP, showed similar substrate selectivity: both SmKS and SmDTC3 synthesized sandaracopimaradiene from normal CDP. Therefore, the diversity of substrate recognition among KSs from other plants was investigated. PpCPS/KS could use normal CDP and syn-CDP as well as ent-CDP as substrate. In contrast, lettuce KS showed high specificity for ent-CDP, and rice KS recognized only ent-CDP. Our studies imply that ancient KS having low substrate specificity has evolved to be specific for ent-CDP to the biosynthesis of gibberellin.
authors.affiliation: *United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Manami
authors.initials: M
authors.lastname: Shimane
authors.affiliation: †Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Ueno
authors.affiliation: †Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Morisaki
authors.affiliation: ‡Department of Biochemistry, Okayama University of Science, Ridaicho, Okayama 700-0005, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Oogami
authors.forename: Masahiro
authors.initials: M
authors.lastname: Natsume
authors.affiliation: ‡Department of Biochemistry, Okayama University of Science, Ridaicho, Okayama 700-0005, Japan.
authors.forename: Ken-Ichiro
authors.initials: K
authors.lastname: Hayashi
authors.affiliation: ‡Department of Biochemistry, Okayama University of Science, Ridaicho, Okayama 700-0005, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nozaki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawaide
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D019669:Embryophyta / Q000201:enzymology; D019143:Evolution, Molecular; D005875:Gibberellins / Q000096:biosynthesis*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010755:Organophosphates / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D032503:Selaginellaceae / Q000201:enzymology / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D005875:Gibberellins; D010755:Organophosphates; D010940:Plant Proteins; C408747:copalyl diphosphate; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A; C105712:ent-kaurene synthetase B