Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis
Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis
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DOI：
10.1104/pp.104.056747
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发表时间：
2005-03-01
期刊：
影响因子：
7.4
通讯作者：
Ayabe, S
中科院分区：
文献类型：
--
作者：
Akashi, T;Aoki, T;Ayabe, S
异黄酮是豆科植物具有生理生态活性的次级代谢产物，具有促进健康的植物雌激素功能。异黄酮是通过从2-羟基异黄烷酮中1，2-消除水而合成的，2-羟基异黄烷酮是具有双羟基骨架的第一个中间体，但这种脱水的细节尚不清楚。我们筛选了表达棘根cDNA文库的反复分级分离的大肠杆菌的提取物，用于将放射性标记的前体转化为芒柄花素（7-羟基-4 '-甲氧基异黄酮）的活性，并分离了2-羟基异黄烷酮转化酶（HID）的克隆。根据表达序列标签文库中的序列信息，从大豆中克隆了另一个HID cDNA。动力学研究表明，G.松果菊HID对2，7-二羟基-4 ′-甲氧基异黄烷酮具有特异性，而大豆HID对4 ′-羟基化和4 ′-甲氧基化的2-羟基异黄烷酮具有更广泛的特异性，反映了每种植物物种中所含的异黄酮的结构。引人注目的是，HID蛋白是一个大的羧酸酯酶家族的成员，其中植物蛋白形成一个单系群，有些被分配防御功能，没有内在的催化活性确定。大豆HID蛋白的定点突变表明，特征性的氧阴离子空穴和催化三联体是必需的，以及微弱的酯酶活性。据我们所知，这些发现代表了植物次生代谢分子进化过程中初级代谢酶的招募的一个新例子。
Isoflavonoids are ecophysiologically active secondary metabolites of the Leguminosae and known for health-promoting phytoestrogenic functions. Isoflavones are synthesized by 1,2-elimination of water from 2-hydroxyisoflavanones, the first intermediate with the isoflavonoid skeleton, but details of this dehydration have been unclear. We screened the extracts of repeatedly fractionated Escherichia coli expressing a Glycyrrhiza echinata cDNA library for the activity to convert a radiolabeled precursor into formononetin (7-hydroxy-4'-methoxyisoflavone), and a clone of 2-hydroxyisoflavanone dehydratase (HID) was isolated. Another HID cDNA was cloned from soybean (Glycine max), based on the sequence information in its expressed sequence tag library. Kinetic studies revealed that G. echinata HID is specific to 2,7-dihydroxy-4'-methoxyisoflavanone, while soybean HID has broader specificity to both 4'-hydroxylated and 4'-methoxylated 2-hydroxyisoflavanones, reflecting the structures of isoflavones contained in each plant species. Strikingly, HID proteins were members of a large carboxylesterase family, of which plant proteins form a monophyletic group and some are assigned defensive functions with no intrinsic catalytic activities identified. Site-directed mutagenesis with soybean HID protein suggested that the characteristic oxyanion hole and catalytic triad are essential for the dehydratase as well as the faint esterase activities. The findings, to our knowledge, represent a new example of recruitment of enzymes of primary metabolism during the molecular evolution of plant secondary metabolism.