Abstract
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.
Highlights
Gibberellins (GAs)1 are one of an important group of phytohormones regulating many aspects of plant growth and development
The product was detected at 6:02 after injection: m/z 272 ([M]ϩ; relative intensity, 32%); m/z 257, m/z 229, and m/z 213. This compound was identified as ent-kaurene by comparing with full mass spectrum of the authentic compound. These results demonstrated that the presence of both CPS and KS activities in mycelia of Phaeosphaeria sp
We report the isolation and characterization of a cDNA encoding the fungal ent-kaurene synthase, fungal-type CPS (FCPS)/KS, from Phaeosphaeria sp
Summary
PCR was performed in a 50-l reaction volume using Expand HF buffer with 1.5 mM MgCl2, 0.25 mM dNTP, 8 M of each primer, 1.75 units of the enzyme (Expand HF, Boehringer Mannheim) and 1 l of double-stranded cDNA solution as described below with the following program: denaturation at 94 °C for 1 min, annealing at 46 °C for 1 min, extension at 72 °C for 2 min (35 cycles), final extension at 72 °C for 7 min. In the case of the cell-free system, each substrate was incubated in 100 l of the S-200 enzyme solution containing Mg2ϩ (5 mM) and uniconazole (1 mM), as ent-kaurene oxidase inhibitor [22], at 30 °C for 30 min. The n-hexane extracts were concentrated under gentle nitrogen flow and injected into a 15-m fused silica capillary column (DB-1, J & W Scientific, Folsom, CA) coupled to an ion-trap GC-MS (GCQ, Thermo Quest, San Jose, CA)
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