Dihydro-p-coumaroyl-CoA dehydrogenase

本课题研究了苹果中含量最高的多酚类物质根皮苷生物合成的关键步骤。根皮苷占苹果叶片可溶性酚类物质的90%以上。如此大量的根皮苷的存在使得苹果独一无二，因为其他物种仅积累非常低的量，并且许多密切相关的物种如梨不能形成根皮素或其葡萄糖基化的相对根皮苷。在过去的十年里，关于根皮素和根皮苷对人类健康有益作用的研究激增，但苹果的生理相关性仍不清楚。一个可能参与抗病性进行了讨论。以前我们已经用苹果叶提取物表明根皮苷的形成是基于三个生物合成步骤：（1）通过脱氢酶从对香豆酰辅酶A形成二氢对香豆酰辅酶A，（2）通过常见的查耳酮合酶进一步形成根皮素，和（3）根皮素在2 '位的葡糖基化。虽然后两个步骤已经被深入研究，但第一个步骤的知识是有限的。这种酶是至关重要的，因为它似乎是使苹果与其他植物相比具有独特性的关键点。在我们之前的FWF项目（P25399-B16）中，我们成功地完成了具有挑战性的纯化过程，并且能够首次从苹果叶中纯化候选酶，该候选酶与对香豆酰辅酶A形成二氢-对香豆酰辅酶A表现出强的酶活性。计划中的后续项目将首次针对苹果叶中这种重要酶的详细表征。结构研究将解决酶机制，如蛋白质结晶和底物，抑制剂/效应物或其他因素的影响。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，用于详细的表征。将测试脱氢酶基因在哪个组织和发育阶段打开或关闭。 基因产物的功能活性将用转基因植物进行测试，其中根皮素的形成将通过塔勒水芹（拟南芥）中的脱氢酶来实现，或在苹果中被禁用。比较不同植物脱氢酶的DNA和蛋白质序列，将有助于从分子水平上了解该酶的结构与活性关系。项目成员由三个团队组成，提供互补的技术和资源：其中一个奥地利团队提供根皮苷生物合成，分子生物学和酶评估方面的知识，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，而德国团队提供基础设施和创造转基因植物的长期经验。

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.