Functional analysis of the two interacting cyclase domains in ent-kaurene synthase from the fungus Phaeosphaeria sp L487 and a comparison with cyclases from higher plants

We report here kinetic analysis and identification of the two cyclase domai ns in a bifunctional diterpene cyclase, Phaeosphaeria ent-kaurene synthase (FCPS/KS). Kinetics of a recombinant FCPS/KS protein indicated that the aff inity for copalyl diphosphate is higher than that for geranylgeranyl diphos phate (GGDP), ent-Kaurene production from GGDP by FCPS/KS was enhanced by t he addition of a plant ent-kaurene synthase (HS) but not by plant CDP synth ase (CPS), suggesting that the rate of ent-kaurene production of FCPS/KS ma y be limited by the KS activity. Site-directed mutagenesis of aspartate-ric h motifs in FCPS/KS indicated that the (DVDD)-D-318 motif near the N termin us and the (DEFFE)-D-656 motif near the C terminus may be part of the activ e site for the CPS and RS reactions, respectively. The other aspartate-rich (DDVLD)-D-132 motif near the N terminus is thought to be involved in both reactions. Functional analysis of the N- and C-terminal truncated mutants r evealed that a N-terminal 59-kDa polypeptide catalyzed the CPS reaction and a C-terminal 66-kDa polypeptide showed RS activity. A 101-kDa polypeptide lacking the first 43 amino acids of the N terminus reduced KS activity seve rely without CPS activity. These results indicate that there are two separa te interacting domains in the 106-kDa polypeptide of FCPS/KS.