Dihydro-p-coumaroyl-CoA dehydrogenase

本课题研究了苹果中普遍存在的多酚类化合物根连苷生物合成的关键步骤。苹果叶中90%以上的可溶性酚类化合物都含有连根茎素。如此高含量的根连素使得苹果独一无二，因为其他物种的根连素积累量很低，而且许多密切相关的物种，如梨，不能形成根连素或其糖基化的相对根连素。在过去的十年里，关于根皮素和根皮苷对人体健康的有益作用的研究激增，但苹果的生理相关性仍不清楚。讨论了对抗病的可能参与。之前我们已经用苹果叶提取物表明，根素的形成是基于三个生物合成步骤：(1)脱氢酶从对coumaroyl- coa形成二氢-对coumaroyl- coa，(2)普通查尔酮合成酶进一步形成根素，(3)根素2 '位的糖基化。虽然后两个步骤已经深入研究，但第一步的知识是有限的。这种酶是至关重要的，因为它似乎是使储存根际素的苹果与其他植物相比独一无二的关键。在我们之前的FWF项目（P25399-B16）中，我们成功地完成了一个具有挑战性的纯化过程，并首次从苹果叶片中纯化出一种候选酶，该酶与对香豆醇辅酶a具有很强的酶活性，形成二氢-对香豆醇辅酶a。计划中的后续项目现在将首次针对苹果叶片中这种重要酶的详细表征。结构研究将解决酶的机制，如蛋白质结晶和底物、抑制剂/效应器或其他因素的影响。脱氢酶的DNA序列将从苹果中分离出来，并转移到细菌中，以产生大量的酶进行详细的表征。它将被测试，在组织和发育阶段脱氢酶基因被打开或关闭。基因产物的功能活性将在转基因植物中进行测试，其中拟南芥（拟南芥）的脱氢酶将使根皮素的形成成为可能，而苹果的脱氢酶将使根皮素的形成失效。比较不同植物脱氢酶的DNA和蛋白质序列，有助于在分子水平上了解该酶的构效关系。项目成员由三个团队组成，他们提供互补的技术和资源：一个奥地利团队提供根连素生物合成、分子生物学和酶评价方面的知识，另一个奥地利团队在蛋白质表征和结晶方面拥有丰富的经验，而德国团队提供转基因植物的基础设施和长期经验。

This project studies the key step in the biosynthesis of phloridzin, which is the prevalent polyphenolic compound in apple. Phloridzin represents more than 90% of the soluble phenolic compounds in apple leaves. The presence of such high amounts of phloridzin makes apple unique since other species accumulate only very low amounts and many closely related species like pear are not able to form phloretin or its glucosylated relative phloridzin. The last decade has seen an explosion of research on the beneficial effects of phloretin and phloridzin for human health but the physiological relevance for apple is still unclear. A possible involvement in disease resistance is discussed. Previously we have shown with apple leaf extracts that phloridzin formation is based on three biosynthetic steps: (1) the formation of dihydro-p-coumaroyl-CoA from p-coumaroyl-CoA by a dehydrogenase, (2) further formation of phloretin by the common chalcone synthase and (3) the glucosylation of phloretin in position 2’. Whereas the last two steps were already intensively studied, the knowledge of the first step is limited. The enzyme is crucial, because it seems to be the key point making the phloridzin-hoarding apple unique in comparison to other plants. In our previous FWF project (P25399-B16) we successfully completed a challenging purification process and were able to purify for the first time a candidate enzyme from apple leaves, which exhibits strong enzyme activity with p-coumaroyl-CoA to form dihydro-p-coumaroyl-CoA. The planned follow-up project will now target the detailed characterization of this important enzyme from apple leaves for the first time. Structural studies will resolve the enzymatic mechanism, such as protein crystallization and effects of substrates, inhibitors/effectors or other factors. The DNA sequence of the dehydrogenase will be isolated from apple and transferred into bacteria to produce large amounts of the enzyme for detailed characterization. It will be tested, in which tissue and developmental stage the dehydrogenase gene is switched on or off. Functional activity of the gene products will be tested with genetically modified plants where phloretin formation will be enabled by the dehydrogenase in thale cress (Arabidopsis) or disabled in apple. Comparison of the DNA and protein sequence of the dehydrogenase from different plant species will give insight to structure-activity relationship of the enzyme on the molecular level. The project members consist of three teams which provide complementary know-how and resources: One of the Austrian teams offers knowledge in phloridzin biosynthesis, molecular biology and enzymatic evaluation, the other Austrian team has profound experience in protein characterization and crystallisation, whereas the German team provides the infrastructure and long-term experience in the creation of transgenic plants.