Dihydro-p-coumaroyl-CoA dehydrogenase: the key enzyme of phloridzin biosynthesis

This project will concentrate on the key step in the biosynthesis of the dihydrochalcone phloridzin, which is the prevalent polyphenol in apple (Malus ssp.). 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 (Pyrus communis) are not able to form phloretin or phloridzin. The last decade has seen an explosion of research on the beneficial effects of phloretin and phloridzin for human health. In contrast, the physiological relevance of phloridzin 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) formation of dihydro-p-coumaroyl-CoA from p-coumaroyl-CoA, (2) formation of phloretin by the common chalcone synthase and (3) glucosylation of phloretin in position 2`. Whereas the last two steps were already intensively studied, the knowledge of the first step is limited. The reaction was demonstrated and characterized biochemically and shows close similarity to the saturation of double bonds of medium chain fatty acids in fatty acid metabolism or phenylpropene aldehydes in lignin biosynthesis. However, attempts to isolate the underlying cDNA clone failed so far due to the novelty of the enzyme. Therefore the dihydro-p-coumaroyl-CoA dehydrogenase (DDH) still remains a puzzle. The enzyme is crucial, because it seems to be the key point making the `phloridzin-hoarding` apple unique in comparison to other plants This project will target the purification and characterization of this important enzyme from apple leaves for the first time. To resolve the enzymatic mechanism of DDH, structural studies are required. We will crystallize both the met DDH and the enzyme in the presence of cofactors. Crystal structures representing complexes of DDH with substrates, inhibitors and various effectors will give us the pictures of various activation states of the enzyme and snapshots of intermediate states throughout the catalytic process. Using a partial amino acid sequence, cDNA clones will be isolated from apple and strawberry leaves and dihydrochalcone formation in Rosaceous species will be studied in detail. The cDNA clones will be heterologously expressed and functional identity will be verified by testing the activity of the recombinant enzymes. Phylogenetic relationships will be analysed. Genomic clones and 5`-flanking regulatory regions encoding isoenzymes as multigene family will be compared. Expression of the isolated genes will be tested for tissue specificity and ontogenetic dependence. Functional activity of the gene products will be tested in vivo by transient and stable introduction into Fragaria vesca. Vectors will be constructed for silencing the HCD in apple and overexpression in pear.

This project studied the key step in the biosynthesis of phloridzin, which is the prevalent polyphenolic compound in apple and beneficial for human health. More than 90% of the soluble phenolic compounds in apple leaves belong to the phloridzin family. 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. We isolated for the first time a novel candidate enzyme from apple leaves, which represents the key step in the biosynthesis of the dihydrochalcones phloretin and phloridzin. The enzyme exhibits strong double bond reductase activity with p-coumaroyl-CoA to form dihydro-p- coumaroyl-CoA, the precursor of dihydrochalcones. We further established and optimized a protocol for the production of the enzyme in microorganisms, which is important in the production of high amounts of purified enzyme, for example. Chalcone synthase (CHS) catalyses the second step in the dihydrochalcone formation. We investigated five different CHS from apple and showed that none of them prefer p- dihydrocoumaroyl-CoA over p-coumaroyl-CoA as substrate which means that CHS is therefore not decisive for whether or not dihydrochalcones are formed in a plant. Hydroxylation of phloretin to 3-hydroxyphloretin is another important unknown step in the dihydrochalcone biosynthesis chain. We demonstrated, that F3H (flavonoid 3-hydroxylase) is not involved in 3-hydroxyphloretin biosynthesis. With purified apple protein extracts and recombinant polyphenol oxidase, in contrast, we demonstrated the specific hydroxylation of phloretin to 3-hydroxyploretin. Additionally we found different dihydrochalcone concentrations and 3-hydroxyphloretin formation as a function of the physiological health status of apple fruits (watercore disease vs. healthy fruits) which could allow the identification of the target dehydrogenase by an additional molecular biological approach. Overall, during the project a major step was taken in the final elucidation of the dihydrochalcone biosynthesis. In addition, new aspects of the substrate specificities of CHS and F3H will shed light on other branches of the flavonoid pathway. In contrast to laborious isolation of single dihydrochalcone compounds from plant material they could be efficiently synthesized with recombinant dehydrogenase obtained within the project. Knowledge of dihydrochalcone formation at the level of genes and enzymes will allow to obtain deeper insight into the health beneficial effects of distinct plants and to optimize diets with respect to dihydrochalcone composition.