Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
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DOI：
10.1073/pnas.96.22.12929
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发表时间：
1999-10-26
影响因子：
11.1
通讯作者：
Winkel-Shirley, B
中科院分区：
文献类型：
--
作者：
Burbulis, IE;Winkel-Shirley, B
黄酮类化合物是由苯丙氨酸和乙酸代谢产生的次生代谢产物，在高等植物中发挥着多种重要功能。过去30年的研究已经支持了一种模型，其中类黄酮代谢由定位于内质网的酶复合物催化[Hrazdina，G. &瓦格纳，G. J.（1985）Arch. Biochem. Biophys. 237，88-100]。为了进一步验证这一模型，我们分析了几个关键的类黄酮生物合成酶之间的直接相互作用，在发展中的拟南芥幼苗。双杂交分析表明，查尔酮合酶，查尔酮异构酶（CHI），和二氢黄酮醇4-还原酶相互作用的方向依赖的方式。亲和层析和免疫沉淀试验进一步证明了拟南芥幼苗裂解液中查尔酮合酶、CHI和黄酮醇3-羟化酶之间的相互作用。这些结果支持的假设，类黄酮酶组装作为一个大分子复合物与多个蛋白质之间的接触。还发现了CHI的翻译后修饰的证据。在代谢工程的背景下，讨论了了解精细酶系统的亚细胞组织的重要性。
Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism that perform a variety of essential functions in higher plants. Studies over the past 30 years have supported a model in which flavonoid metabolism is catalyzed by an enzyme complex localized to the endoplasmic reticulum [Hrazdina, G. & Wagner, G. J. (1985)Arch. Biochem. Biophys. 237, 88-100]. To test this model further we assayed for direct interactions between several key flavonoid biosynthetic enzymes in developing Arabidopsis seedlings. Two-hybrid assays indicated that chalcone synthase, chalcone isomerase (CHI), and dihydroflavonol 4-reductase interact in an orientation-dependent manner. Affinity chromatography and immunoprecipitation assays further demonstrated interactions between chalcone synthase, CHI, and flavonol 3-hydroxylase in lysates from Arabidopsis seedlings. These results support the hypothesis that the flavonoid enzymes assemble as a macromolecular complex with contacts between multiple proteins. Evidence was also found for posttranslational modification of CHI. The importance of understanding the subcellular organization of elaborate enzyme systems is discussed in the context of metabolic engineering.