EXPRESSION CLONING OF A GIBBERELLIN 20-OXIDASE, A MULTIFUNCTIONAL ENZYME INVOLVED IN GIBBERELLIN BIOSYNTHESIS
EXPRESSION CLONING OF A GIBBERELLIN 20-OXIDASE, A MULTIFUNCTIONAL ENZYME INVOLVED IN GIBBERELLIN BIOSYNTHESIS
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DOI：
10.1073/pnas.91.18.8552
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发表时间：
1994-08-30
影响因子：
11.1
通讯作者：
GRAEBE, JE
中科院分区：
文献类型：
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作者：
LANGE, T;HEDDEN, P;GRAEBE, JE
在赤霉素（GAs）的生物合成途径中，碳-20被氧化去除，生成C-19-GAs，其中包括具有生物活性的植物激素。我们报道了一个编码GA 20-氧化酶[赤霉素，2-氧葡萄糖酸酯：氧氧化还原酶（20-羟基化，氧化）EC 1.14.11的cDNA克隆的分离。-]通过筛选发育中的南瓜（Cucurbita maxima L.）子叶的cDNA文库来表达该酶。当子叶或胚乳的mRNA在体外用兔网织细胞裂解物翻译时，产物具有GA(12) 20氧化酶活性。通过胰酶消化从胚乳中纯化的GA - 20氧化酶所释放的肽的氨基酸序列，建立了一种多克隆抗血清。利用子叶mRNA构建了lambda gt11 cDNA表达文库，并用抗血清进行筛选。通过在单个噬菌体斑块中显示GA(12) 20氧化酶活性，证实了阳性克隆的身份。重组蛋白催化GA（12）向GA（25）和GA（53）向GA（17）的三步转化，并由GA（23）、GA（24）和GA（19）分别生成C-19-GAs、GA(1)、GA(9)和GA（20）。cDNA插入片段的核苷酸序列包含1158 nt的开放阅读框，编码386个氨基酸残基的蛋白。预测的M(r)（43,321）和pI（5.3）与天然GA 20氧化酶的实验结果相似。此外，衍生的氨基酸序列包括从N端获得的序列和从天然酶获得的两个色氨酸肽。它还包含在一组非血红素含铁双加氧酶中高度保守的区域。
In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C-19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA(12) 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA(12) 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA(12) to GA(25) and of GA(53) to GA(17), as well as the formation of the C-19-GAs, GA(1), GA(9), and GA(20), from their respective aldehyde precursors, GA(23), GA(24), and GA(19). The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases.