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.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Gibberellins/biosynthesis , Plant Proteins/metabolism , Alkyl and Aryl Transferases/genetics , Cloning, Molecular , Embryophyta/enzymology , Evolution, Molecular , Nuclear Magnetic Resonance, Biomolecular , Organophosphates/metabolism , Plant Proteins/genetics , Selaginellaceae/enzymology , Selaginellaceae/genetics , Stereoisomerism , Substrate SpecificityABSTRACT
Polyphosphate kinase (PPK), which can regenerate ATP from ADP, was utilized in the mevalonate-dependent enzymatic synthesis of amorphadiene. The activity of PPK, cloned from Escherichia coli, was determined by (31)P-NMR. The yield from the PPK-catalyzed synthesis was 25%, 2.5 times higher than that without PPK. The (31)P-NMR analysis of the final reaction mixture indicated no accumulation of intermediates.
