title: Identification and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase involved in dihydrochalcone formation in Malus×domestica Borkh.
abstract: The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages. Many of the health beneficial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Arabidopsis alkenal double bond reductases. Herein described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3μM, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the α,β-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds.
authors.affiliation: Institute of Biological Chemistry, Washington State University, PO Box 646340, Pullman, WA 99164-6340, USA; NeweYaar Research Center, Agriculture Research Organization, PO Box 1021, Ramat Yishay 30095, Israel.
authors.forename: Mwafaq
authors.initials: M
authors.lastname: Ibdah
authors.affiliation: Institute of Biological Chemistry, Washington State University, PO Box 646340, Pullman, WA 99164-6340, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Berim
authors.affiliation: Fondazione Edmund Mach, Centro Ricerca e Innovazione, Department of Food Quality and Nutrition, Via E. Mach, 1 - 38010 San Michele all'Adige (TN), Italy.
authors.forename: Stefan
authors.initials: S
authors.lastname: Martens
authors.affiliation: Fondazione Edmund Mach, Centro Ricerca e Innovazione, Department of Food Quality and Nutrition, Via E. Mach, 1 - 38010 San Michele all'Adige (TN), Italy.
authors.forename: Andrea Lorena Herrera
authors.initials: AL
authors.lastname: Valderrama
authors.affiliation: Fondazione Edmund Mach, Centro Ricerca e Innovazione, Department of Food Quality and Nutrition, Via E. Mach, 1 - 38010 San Michele all'Adige (TN), Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Palmieri
authors.affiliation: NeweYaar Research Center, Agriculture Research Organization, PO Box 1021, Ramat Yishay 30095, Israel.
authors.forename: Efraim
authors.initials: E
authors.lastname: Lewinsohn
authors.affiliation: Institute of Biological Chemistry, Washington State University, PO Box 646340, Pullman, WA 99164-6340, USA. Electronic address: gangd@wsu.edu.
authors.forename: David R
authors.initials: DR
authors.lastname: Gang
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D017360:Arabidopsis / Q000378:metabolism; D047188:Chalcones / Q000378:metabolism*; D019976:Cloning, Organism; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D035264:Flowers / Q000235:genetics / Q000378:metabolism; D027845:Malus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D059808:Polyphenols / Q000737:chemistry
chemical_list: D000214:Acyl Coenzyme A; D047188:Chalcones; D003374:Coumarins; D059808:Polyphenols; C119167:feruloyl-CoA; C058644:4-coumaroyl-coenzyme A; D009249:NADP; D010088:Oxidoreductases; C015812:dihydrochalcone
keywords: Apple; Dihydrochalcones; Feruloyl-CoA; Hydroxycinnamoyl-CoA double bond reductase; Malus×domestica; Rosaceae; p-Coumaroyl-CoA; p-Dihydrocoumaroyl-CoA