ENT-KAURENE SYNTHASE FROM THE FUNGUS PHAEOSPHAERIA SP L487 - CDNA ISOLATION, CHARACTERIZATION, AND BACTERIAL EXPRESSION OF A BIFUNCTIONAL DITERPENE CYCLASE IN FUNGAL GIBBERELLIN BIOSYNTHESIS

ent-Kaurene is the first cyclic diterpene intermediate of gibberellin biosynthesis in both plants and fungi, In plants, ent-kaurene is synth esized from geranylgeranyl diphosphate via copalyl diphosphate in a tw o-step cyclization catalyzed by copalyl diphosphate synthase and ent-k aurene synthase. A cell-free system of the fungus Phaeosphaeria sp, L4 87 converted labeled geranylgeranyl diphosphate to ent-kaurene, A cDNA fragment, which possibly encodes copalyl diphosphate synthase, was is olated by reverse transcription-polymerase chain reaction using degene rate primers based on the consensus motifs of plant enzymes, Translati on of a full-length cDNA sequence isolated from the fungal cDNA librar y revealed an open reading frame for a 106-kDa polypeptide, The deduce d amino acid sequence shared 24 and 21% identity with maize copalyl di phosphate synthase and pumpkin ent-kaurene synthase, respectively, A f usion protein produced by expression of the cDNA in Escherichia coli c atalyzed the two-step cyclization of geranylgeranyl diphosphate to ent -kaurene, Amo-1618 completely inhibited the copalyl diphosphate syntha se activity of the enzyme at 10(-6) M, whereas it did not inhibit the ent-kaurene synthase activity even at 10(-4) M, These results indicate that the fungus has a bifunctional diterpene cyclase that can convert geranylgeranyl diphosphate into ent-kaurene. They may be separate cat alytic sites for the two cyclization reactions.