Ent-kaurene is a diterpene hydrocarbon found in both higher plants and bryophytes. Ent-kaurene is biosynthesized from geranylgeranyl diphosphate (GGDP) by ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) in higher plants. In bryophytes, however, one bifunctional enzyme CPS/KS catalyzes the two-step cyclization from GGDP to ent-kaurene. We have shown the function of two CPS/KSs, one is from moss Physcomitrella patens which converts GGDP to ent-kaurene and ent-16αhydroxykaurene. Another CPS/KS is from liverwort Jungermannia subulata which converts GGDP to only ent-kaurene. We herein report the amino acid residues in CPS/KS responsible for the final cyclization step from ent-copalyl diphosphate to ent-kaurene. Recombinant proteins of two CPS/KSs were synthesized using in vitro protein synthesis kit. A chimeric protein with PpCPS/KS on N-terminal domain and JsCPS/KS on C-terminal domain catalyzed the cyclization reaction of GGDP to only ent-kaurene. This result suggested that the amino acid residues responsible for carbocation-quenching mechanism with H_2O may be located in the C-terminal region. To determine the target amino acid residues to cause hydroxylation, another type of chimeric proteins was synthesized, by substituting a part of amino acids in PpCPS/KS with JsCPS/KS. The protein substituted 48 amino acids in PpCPS/KS with JsCPS/KS produced only ent-kaurene from GGDP.
