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 synthesized from geranylgeranyl diphosphate via copalyl diphosphate in a two-step cyclization catalyzed by copalyl diphosphate synthase and ent-kaurene synthase. A cell-free system of the fungus Phaeosphaeria sp, L487 converted labeled geranylgeranyl diphosphate to ent-kaurene, A cDNA fragment, which possibly encodes copalyl diphosphate synthase, was isolated by reverse transcription-polymerase chain reaction using degenerate primers based on the consensus motifs of plant enzymes, Translation of a full-length cDNA sequence isolated from the fungal cDNA library revealed an open reading frame for a 106-kDa polypeptide, The deduced amino acid sequence shared 24 and 21% identity with maize copalyl diphosphate synthase and pumpkin ent-kaurene synthase, respectively, A fusion protein produced by expression of the cDNA in Escherichia coli catalyzed the two-step cyclization of geranylgeranyl diphosphate to ent-kaurene, Amo-1618 completely inhibited the copalyl diphosphate synthase 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 catalytic sites for the two cyclization reactions.