Dihydro-p-coumaroyl-CoA dehydrogenase

二氢对香豆酰辅酶A脱氢酶

• 批准号: 423978791
• 负责人: Professor Dr. Henryk Flachowsky
• 金额: --
• 依托单位: Institut für Züchtungsforschung an Obst
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/423978791?language=en
• 关键词: Dihydro; coumaroyl; CoA; dehydrogenase

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.

本课题对苹果中普遍存在的多酚类化合物根茎苷的生物合成关键步骤进行了研究。根茎苷占苹果叶片中90%以上的可溶性酚类化合物。这种高含量根茎苷的存在使苹果独一无二，因为其他物种只积累了很少的量，而且许多密切相关的物种，如梨，无法形成根霉素或其糖基化的相对根茎苷。在过去的十年里，关于根瘤素和根茎苷对人类健康有益的研究出现了爆炸性的增长，但对苹果的生理相关性仍不清楚。讨论了参与抗病的可能性。以前，我们已经用苹果叶提取物证明根茎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-CoA形成二氢-对-香豆酰-CoA，(2)通过普通查尔酮合成酶进一步形成根苷，(3)在2‘位糖基化根茎苷。虽然后两个步骤已经得到了深入的研究，但对第一个步骤的了解有限。这种酶是至关重要的，因为它似乎是使囤积根瘤菌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，并首次能够从苹果叶片中纯化出一种候选酶，该酶具有很强的酶活性，与p-香豆酰辅酶A形成二氢-p-香豆酰辅酶A。计划中的后续项目现在将首次以苹果叶片中这种重要酶的详细特征为目标。结构研究将解决酶机制，如蛋白质结晶和底物、抑制剂/效应剂或其他因素的影响。脱氢酶的DNA序列将从苹果中分离出来，并转移到细菌中，以生产大量的酶，以便进行详细的鉴定。它将被测试，在哪个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，在转基因植物中，根黄素的形成将被拟南芥(拟南芥)中的脱氢酶激活或在苹果中被禁用。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脱氢酶的构效关系。项目成员由三个团队组成，提供互补的技术诀窍和资源：其中一个奥地利团队提供根霉毒素生物合成、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深厚的经验，而德国团队提供转基因植物创造方面的基础设施和长期经验。