Dihydro-p-coumaroyl-CoA dehydrogenase

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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.