The gibberellin 20-oxidase of Gibberella fujikuroi is a multifunctional monooxygenase
The gibberellin 20-oxidase of Gibberella fujikuroi is a multifunctional monooxygenase
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DOI：
10.1074/jbc.m201651200
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发表时间：
2002-06-14
影响因子：
4.8
通讯作者：
Hedden, P
中科院分区：
文献类型：
--
作者：
Tudzynski, B;Rojas, MC;Hedden, P
赤霉素（GA）生物合成的基因聚集在真菌藤黑赤霉中。除了编码 GA 特异性香叶基香叶基二磷酸合酶和双功能对映柯巴基二磷酸/对映贝壳杉烯合酶的基因外，该簇还包含四个细胞色素 P450 单加氧酶基因（P450-1、-2、-3、-4）。最近发现P450-4和P450-1编码多功能酶，分别催化从对映贝壳杉烯到对映贝壳杉烯酸的三个氧化步骤和从对映贝壳杉烯酸到GA(14)的四个氧化步骤。在这里，我们描述了通过基因破坏和在缺乏整个 GA 生物合成基因簇的突变体中表达该基因来对 P450-2 基因进行功能分析。突变体中的 P450-2 通过插入一大段 DNA 而失活，积累了 GA(14)，并且缺乏生物合成更高级的代谢物，表明该基因编码 20-氧化酶。这通过在不存在其他 GA 生物合成基因和同位素标记底物的情况下孵育含有 P450-2 的品系来证实。 P450-2基因产物将3β-羟基化中间体GA(14)及其非羟基化类似物GA(12)分别氧化为GA(4)和GA(9)。 P450-2 的表达受到培养基中大量氮的抑制，但不受生物合成高级 GA 存在的影响，即没有反馈调节的证据。事实上，G. fujikuroi 中的 GA 20-氧化酶是一种细胞色素 P450 单加氧酶，而不是植物中的 2-酮戊二酸依赖性双加氧酶，加上基因表达调控的显着差异，进一步证明了植物和真菌中 GA 生物合成途径的独立进化。
The genes for gibberellin (GA) biosynthesis are clustered in the fungus Gibberella fujikuroi. In addition to genes encoding a GA-specific geranylgeranyl diphosphate synthase and a bifunctional ent-copalyl diphosphate/ent-kaurene synthase, the cluster contains four cytochrome P450 monooxygenase genes (P450-1, -2, -3, -4). Recently it was shown that P450-4 and P450-1 encode multifunctional enzymes catalyzing the three oxidation steps from ent-kaurene to ent-kaurenoic acid and the four oxidation steps from ent-kaurenoic acid to GA(14), respectively. Here we describe the functional analysis of the P450-2 gene by gene disruption and by expressing the gene in a mutant that lacks the entire GA biosynthesis gene cluster. Mutants in which P450-2 is inactivated by the insertion of a large piece of DNA accumulated GA(14) and lacked biosynthetically more advanced metabolites, indicating that the gene encodes a 20-oxidase. This was confirmed by incubating lines containing P450-2 in the absence of the other GA biosynthesis genes with isotopically labeled substrates. The P450-2 gene product oxidized the 3beta-hydroxylated intermediate, GA(14), and its non-hydroxylated analogue GA(12) to GA(4) and GA(9), respectively. Expression of P450-2 is repressed by high amounts of nitrogen in the culture medium but is not affected by the presence of biosynthetically advanced GAs, i.e. there is no evidence for feedback regulation. The fact that the GA 20-oxidase is a cytochrome P450 monooxygenase in G. fujikuroi and not a 2-oxoglutarate-dependent dioxygenase as in plants, together with the significant differences in regulation of gene expression, are further evidence for independent evolution of the GA biosynthetic pathways in plants and fungi.