Cloning and expression of two novel aldo-keto reductases from Digitalis purpurea leaves.
The aldo-keto reductase (AKR) superfamily comprises proteins that catalyse mainly the reduction of carbonyl groups or carbon-carbon double bonds of a wide variety of substrates including steroids. Such types of reactions have been proposed to occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis plants. Two cDNAs encoding leaf-specific AKR proteins (DpAR1 and DpAR2) were isolated from a D. purpurea cDNA library using the rat Delta4-3-ketosteroid 5beta-reductase clone. Both cDNAs encode 315 amino acid proteins showing 98.4% identity. DpAR proteins present high identities (68-80%) with four Arabidopsis clones and a 67% identity with the aldose/aldehyde reductase from Medicago sativa. A molecular phylogenetic tree suggests that these seven proteins belong to a new subfamily of the AKR superfamily. Southern analysis indicated that DpARs are encoded by a family of at most five genes. RNA-blot analyses demonstrated that the expression of DpAR genes is developmentally regulated and is restricted to leaves. The expression of DpAR genes has also been induced by wounding, elevated salt concentrations, drought stress and heat-shock treatment. The isolated cDNAs were expressed in Escherichia coli and the recombinant proteins purified. The expressed enzymes present reductase activity not only for various sugars but also for steroids, preferring NADH as a cofactor. These studies indicate the presence of plant AKR proteins with ketosteroid reductase activity. The function of the enzymes in cardenolide biosynthesis is discussed.
Gavidia
Isabel
I
Center of Plant Molecular Biology (ZMBP), University of Tübingen, Germany. Isabel.gavidia@uv.es
Pérez-Bermúdez
Pedro
P
Seitz
H Ulrich
HU
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000302:isolation & purification; D004070:Digitalis / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D000455:Medicago sativa / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase