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J Biol Chem, Vol. 275, Issue 4, 2276-2280, January 28, 2000

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^*

Hiroshi Kawaide, Takeshi Sassa^§, and Yuji Kamiya

From the Laboratory for Plant Hormone Function, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan and the ^§ Department of Bioresources, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan

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 ³¹⁸DVDD motif near the N terminus and the ⁶⁵⁶DEFFE motif near the C terminus may be part of the active site for the CPS and KS reactions, respectively. The other aspartate-rich ¹³²DDVLD 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.

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