Identification and functional analysis of bifunctional ent‐kaurene synthase from the moss Physcomitrella patens

ent ‐Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent ‐kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent ‐kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent ‐kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent ‐kaurene synthase (PpCPS/KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101‐kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent ‐kaurene skeleton from GGDP. PpCPS/KS has two aspartate‐rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent ‐kaurene and ent ‐16α‐hydroxykaurene).