49(P01) Phaeosphaeria sp. L487株による糸状菌由来のジベレリン生合成酵素遺伝子に関する研究(ポスター発表の部)

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We are studying gibberellin (GA) biosynthesis of the fungus Phaeosphaeria sp. L487 that has GA 3β-hydroxylation step similar to that in plants. (1) Elucidation of late stages of plant-like GA biosynthetic pathway in Phaeosphaeria sp. L487 We identified GA_1, GA_4, GA_9, GA_<12>, GA_<15>, GA_<20>, GA_<24> and GA_<25> from extracts of Phaeosphaeria's culture filtrate. GA_1 was accumulated in the culture broth at 3 weeks after inoculation (GA_1> 100 mg litre^<-1>). To deremine late stages of the GA biosynthetic pathway in Phaeosphaeria, feeding studies using isotope-labeled GA intermediates were carried out. The fungus metabolized GA_<12>-aldehyde to GA_1 via GA_<12> → GA_<15> → GA_<24>→GA_9→GA_4 and GA_<20>. The 3β-hydroxylation, which is the activation step of endogenous GAs in plants, occurred at steps on GA_9 and GA_<20> to produce GA_4 and GA_1, respectively. (2) Molecular cloning, characterization and identification of catalytic domains of ent-kaurene synthase from Phaeosphaeria sp. L487 ent-Kaurene is the first cyclic intermediate of GA biosynthesis and is synthesized from geranylgeranyl diphosphate (GGDP) via copalyl diphosphate (CDP) in both fungi and plants. In order to clone a cDNA encoding ent-kaurene synthase (KS) from the fungus Phaeosphaeria sp. L487, degenerate primers based on consensus motifs of plant copalyl diphosphate synthase (CPS) and other diterpene cyclases were used to amplify a cDNA fragment encoding the fungal CPS homologue. A full-length cDNA was obtained from the fungal cDNA library and has an open reading frame encoding 946 amino acids with 106-kDa. A fusion protein produced by expression of the cDNA in E. coli catalyzed the two-step cyclization from GGDP to ent-kaurene. Thus, it revealed that ent-kaurene synthase from the fungus is a bifunctional diterpene cyclase. To determine active sites of the enzyme, site-directed mutagenesis was carried out in the aspartatelglutamate-rich motifs, which are thought to be involved in CPS/KS activities. Mutation of Asp132 to Ala132 decreased CPS activity. Mutation of Asp320 to Ala320 had no CPS activity, whereas it increased KS activity. In contrast, mutation of Asp656 to Ala656 had no KS activity. The catalytic domains for two enzyme activities, CPS and KS, on the polypeptide were identified by functional analysis of truncated proteins prepared from deletion clones from the cDNA. A 63-Kda protein translated from the N-terminus of wild type (amino acids 1-568, CTP-2) showed CPS activity, whereas a 59-KDa protein (amino acids 1-530, CTP-1) did not have CPS activity. In contrast, a 66-KDa protein lacking the N-terminus of wild type (359-946 amino acid residues, NTP-3) showed KS activity. A 101-KDa and a 63-KDa protein truncated 44 and 381 amino acid residues from the N-terminus of wild type, respectively, did not have any functions for CPS and KS activity. In conclusion, the fungal ent-kaurene synthase has separate CPS and KS domains fused tandem for the two cyclization reactions.
天然有機化合物討論会の論文
1998-08-31

49(P01) Phaeosphaeria sp. L487株による糸状菌由来のジベレリン生合成酵素遺伝子に関する研究(ポスター発表の部)

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