Identification of the single amino acid involved in quenching the ent‐kauranyl cation by a water molecule in ent‐kaurene synthase of Physcomitrella patens

ent‐Kaurene is a tetracyclic diterpene hydrocarbon and a biosynthetic intermediate of the plant hormone gibberellins. In flowering plants, ent‐kaurene is biosynthesized from geranylgeranyl diphosphate (GGDP) by two distinct cyclases, ent‐copalyl diphosphate synthase (CPS) and ent‐kaurene synthase (KS). Recently, the moss Physcomitrella patens ent‐kaurene biosynthetic gene was cloned and functionally characterized. The bifunctional ent‐kaurene synthase [P. patens CPS/KS (PpCPS/KS)] produces both ent‐kaurene and 16α‐hydroxy‐ent‐kaurane from GGDP via ent‐copalyl diphosphate. Here, we cloned and analyzed the function of a cDNA encoding bifunctional ent‐kaurene synthase from the liverwort Jungermannia subulata [J. subulata CPS/KS (JsCPS/KS)]. JsCPS/KS catalyzes the cyclization reaction of GGDP to produce ent‐kaurene but not 16α‐hydroxy‐ent‐kaurane, even though the PpCPS/KS (881 amino acids) and JsCPS/KS (886 amino acids) sequences share 60% identity. To determine the regions and amino acids involved in 16α‐hydroxy‐ent‐kaurane formation, we analyzed the enzymic functions of JsCPS/KS and PpCPS/KS chimeric proteins. When the C‐terminal region of PpCPS/KS was exchanged with the JsCPS/KS C‐terminal region, the chimeric cyclases produced only ent‐kaurene. The replacement of PpCPS/KS Ala710 with Met or Phe produced a JsCPS/KS‐type cyclase that converted GGDP to ent‐kaurene as the sole product. In contrast, replacing Ala710 with Gly, Cys or Ser did not affect the PpCPS/KS product profile as much as replacement of Cys of JsCPS/KS by Ala. Thus, the hydrophobicity and size of the side chain residue at the PpCPS/KS amino acid 710 is responsible for quenching the ent‐kauranyl cation by the addition of a water molecule.