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 domains in a bifunctional diterpene cyclase, Phaeosphaeria ent-kaurene synthase (FCPS/KS). Kinetics of a recombinant FCPS/KS protein indicated that the affinity for copalyl diphosphate is higher than that for geranylgeranyl diphosphate (GGDP). ent-Kaurene production from GGDP by FCPS/KS was enhanced by the addition of a plant ent-kaurene synthase (KS) but not by plant CDP synthase (CPS), suggesting that the rate of ent-kaurene production of FCPS/KS may be limited by the KS activity. Site-directed mutagenesis of aspartate-rich motifs in FCPS/KS indicated that the (318)DVDD motif near the N terminus and the (656)DEFFE motif near the C terminus may be part of the active site for the CPS and KS reactions, respectively. The other aspartate-rich (132)DDVLD motif near the N terminus is thought to be involved in both reactions. Functional analysis of the N- and C-terminal truncated mutants revealed that a N-terminal 59-kDa polypeptide catalyzed the CPS reaction and a C-terminal 66-kDa polypeptide showed KS activity. A 101-kDa polypeptide lacking the first 43 amino acids of the N terminus reduced KS activity severely without CPS activity. These results indicate that there are two separate interacting domains in the 106-kDa polypeptide of FCPS/KS.
Kawaide
H
H
Laboratory for Plant Hormone Function, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan. hkawaide@postman.riken.go.jp
Sassa
T
T
Kamiya
Y
Y
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000201:enzymology*
D017931:DNA Primers; D019883:Alkyl and Aryl Transferases; C105712:ent-kaurene synthetase B