Institute of Chemical Engineering
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Dihydro-p-coumaroyl-CoA-dehydrogenase

Projektnummer P 25399 (FWF Einzelprojekt)

 

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.

Biosynthesis of phloridzin in Malus ssp. DDH: dihydro-p-coumaroyl-CoA dehydrogenase, CHS: chalcone synthase, DCH2’GT: dihydrochalcone 2’-O-glucosyltransferase

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.

 

Funding:

FWF, Projectnumber P 25399-B16

Duration:

2013 – 2017

 

Leader and Contact:

Privatdoz. Dipl.-Ing. Dr.techn. Heidrun Halbwirth

Executive Senior Researcher

Projektass.(FWF) Dipl.-Chem. Dr.rer.nat. Christian Molitor

Co-workers:

Projektass.(FWF) Dipl.-Ing. Dr.rer.nat. Christian Haselmair-Gosch