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 synthase from the fungus Phaeosphaeria sp. L487 - cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase in fungal gibberellin biosynthesis
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DOI：
10.1074/jbc.272.35.21706
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发表时间：
1997-08-29
影响因子：
4.8
通讯作者：
Kamiya, Y
中科院分区：
文献类型：
--
作者：
Kawaide, H;Imai, R;Kamiya, Y
ent-Kaurene是植物和真菌中赤霉素生物合成的第一环二萜中间体。在植物中，ent-Kaurene是由二磷酸香叶香叶基经二磷酸共丙醇经二磷酸共丙醇催化两步环化合成的，共丙醇二磷酸合酶和ent-Kaurene合酶催化。真菌Phaeosphaeria sp， L487的无细胞系统将标记的geranylgeranyl二磷酸转化为对-丁香烯，利用退化引物，根据植物酶的一致基序，通过逆转录-聚合酶链反应分离出一个可能编码copalyl二磷酸合成酶的cDNA片段，并从真菌cDNA文库中分离出全长cDNA序列，翻译出一个106 kda的开放阅读框。在大肠杆菌中表达cDNA产生的融合蛋白可催化香叶香叶二磷酸两步环化成对-kaurene，在10(-6)M处，Amo-1618完全抑制该酶的共丙二磷酸合成酶活性，而在10(-4)M处，Amo-1618对对-kaurene合成酶活性无抑制作用。这些结果表明，该真菌具有双功能二萜环化酶，可将香叶基二磷酸转化为对karen。它们可能是两个环化反应的单独催化位点。
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.