Dihydroflavonol 4-reductase

Dihydroflavonol 4-reductase (DFR) catalyzes the stereospecific conversion of 2R,3R)-trans-DHFs to the respective (2R,35, 45)-flavan-2,3-traKi-3,4-cA-diols (leucoanthocyanidins) through a NADPH-dependent reduction at the 4-carbonyl. DNA sequences for DFR were first identified from A. majus and Z. mays, and the identity of the Z. mays sequence confirmed by in vitro transcription and translation of the cDNA and assay of the resultant protein. DNA sequences have now been cloned from many species, with the size of the predicted protein averaging about 38kDa. Stereospecificity to (2R,3R)-dihydroquercetin (DHQ) has been shown for some recombinant DFR proteins.  

DFR belongs to the single-domain-reductase/epimerase/dehydrogenase (RED) protein family, which has also been termed the short chain dehydrogenase/reductase (SDR) superfamily. This contains other flavonoid biosynthetic enzymes, in particular the anthocyanidin reductase (ANR), leucoanthocyanidin reductase (EAR), isoflavone reductase (IFR), and vestitone reductase (VR), as well as mammalian, bacterial, and other plant enzymes.  

The preference shown by DFR toward the three common DHFs varies markedly between species, with some enzymes showing little or no activity against dihydrokaempferol (DHK) and others showing preference toward dihydromyricetin (DHM). In particular, DFR in Cymbidium hybrida (cymbidium orchids), L. esculentum. Petunia, and Vaccinium macrocarpon (cranberry) cannot efficiently reduce so that pelargonidin-based anthocyanins 

Some species contain a closely related enzyme activity to DFR that can act on tlavanones, termed the flavanone 4-reductase (FNR), which may represent a variant DFR form. This is discussed in more detail in Section 3.4.7. 5-Deoxyleucoanthocyanidin compounds are known to occur in legumes, and analysis of two recombinant DFR proteins (MtDFRl and MtDFR2) from Medicago truncatula (barrel medic) has found activity on the 5-deoxyDHF substrates fustin and dihydrorobinetin. Indeed, fustin was the preferred substrate of both recombinant enzymes. MtDFRl and MtDFR2 showed distinct enzyme characteristics, and overexpression of MtDFRl but not MtDFR2 promoted anthocyanin biosynthesis in flowers of N. tabacum. 

Substrate specificity between DHK, DHQ, and DHM appears, based on chimeric DFR proteins formed using the P. hybrida and Gerbera hybrida sequences, to locate to a 26 amino acid region that may be the binding pocket for the B-ring, and as little as one amino acid change in this region can alter the specificity of the enzyme.  

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Table 6.1 Abbreviations BAN, BANYULS bHLH, basic helix-loop-helix CHS, chalcone synthase CHI, chalcone isomerase DFR, dihydroflavonol reductase F3H, flavonol 3-hydroxylase F3 H, flavonoid 3 -hydroxylase FLS, flavonol synthase icx, increased chalcone synthase expression LDOX, leucoanthocyanidin dioxygenase LCR, leucoanthocyanidin reductase MATE, multidrug and toxic compound extrusion NR, not yet reported tt, transparent testa ttg, transparent testa glabrous the WD40 and WRKY transcription factors are named for conserved amino acid sequences within these proteins. PC = personal communication.

Carron, T.R., Robbins, M.P., and Morris, P., Genetic modification of condensed tannin biosynthesis in Lotus corniculatus. 1. Heterologous antisense dihydroflavonol reductase down-regulates tannin accumulation in hairy root cultures. Theor. Appl. Genet., 87, 153, 1994. 

Bavage, A.D. et al.. Expression of an Antirrhinum dihydroflavonol reductase gene results in changes in condensed tannin structure and accumulation in root cultures of Lotus corniculatus (bird s foot trefoil). Plant Mol Biol, 35, 443, 1997. 

Robbins, M.P. et al.. Genetic manipulation of condensed tannins in higher plants. II. Analysis of birdsfoot trefoil plants harboring antisense dihydroflavonol reductase constructs. Plant Physiol, 116, 1133, 1998. 

Fig. (1). Schematic view of some branches of phenylpropanoid metabolism. Solid arrows indicate enzymatic reactions with the respective enzyme indicated on the right. PAL, phenylalanine ammonia-lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA ligase CHS, chalcone synthase CF1, chalcone flavavone isomerase F3H, flavanone 3-hydroxylase DFR, dihydroflavonol reductase CHR, chalcone reductase. Broken arrows indicate metabolic branches towards several classes of phenylpropanoids, or several subsequent enzymatic steps. In some cases the enzymes indicated are also involved in other reactions, not shown.

Scheme 1.1 Pathway for the biosynthesis of the major classes of flavonoids. 1, Chalcone synthase 2, chalcone isomerase 3, flavone synthase 4, flavanone 3-hydroxylase 5, flavonol synthase 6, dihydroflavonol reductase 7, anthocyanidin synthase 8, anthocyanidin glucosyltransferase 9, chalcone-ketide reductase 10, chalcone isomerase 11, isoflavone synthase 12, isoflavone 2 -hydroxylase 13, isoflavone reductase 14, pterocarpan synthase 15, pterocarpan 6a-hydroxylase 16, prenyltransferase 17, prenylcyclase.

MURRAY, J. R., HACKETT, W. P., Dihydroflavonol reductase activity in relation to differential anthocyanin accumulation in juvenile and mature phase Hedera helix L., Plant Physiol., 1991,97,343-351. 

Fig. 11.1 Simplified diagram of the flavonoid biosynthetic pathway, starting with the general phenylpropanoid metabolism and leading to the main types of flavonoids. Only a few examples are illustrated of the large variety of flavonoids that arise through modification at different positions (not indicated or shown as R). Enzymes catalysing some key reactions are indicated by the following abbreviations PAL, phenylalanine ammonia-lyase CHS, chalcone synthase CHI, chalcone isomerase DFR, dihydroflavonol reductase F3H, flavanone 3-hydroxylase F3 5 H, flavonoid 3 5 -...

Scheme 1 Schematic representation ofthe biosynthesis pathways of anthocyanins and proanthocyanidins. CHS, chalcone synthase FS, flavanone synthase F3H, flavanone-3-hydroxylase FLS, flavone synthase DFR, dihydroflavonol reductase LAR, leucoanthocyanidin reductase ANS, anthocyanidin synthase ANR, anthocyanidin reductase.

Metabolically, anthocyanins are built up from the dihydroflavonols by means of a reduction of C4, catalyzed by the dihydroflavonol reductase, which leads to the flavan-2,3-trans-3,4-cis-diols, which are intermediates of proanthocyanidins and anthocyanidins. However, despite all the data in this direction, it has not been possible to obtain in vitro the transformation of leucoanthocyanidins in anthocyanins [33],... 

PETERS, D.J., CONSTABEL, C.P., Molecular analysis of herbivore-induced condensed tannin synthesis cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides). Plant J., 2002,32, 701-712. 

An actual industrial application promoted by Suntory Ltd. (Japan) and Florigene Ply Ltd. (Australia) is the exploitation of P450s involved in biosynthesis of delphinidin-type anthocyanins for the production of roses and carnations with nonnatural colors that cannot be achieved by classical breeding [428]. Expression of the flavo-noid 3, 5 -hydroxylase (F3, 5 -H CYP75A) and dihydroflavonol reductase (DFR) from Petunia in DFR-deficient variants led to an exclusive accumulation of delphinidin derivatives and a significant color shift towards blue (Fig. 8.22) [429]. 

Initiating another branch of the flavonoid pathway, C4 of dihydroflavonol 16 can be reduced from a carbonyl group to a hydroxyl group by the oxidoreductase enzyme dihydroflavonol reductase (DFR), producing leucoanthocyanidins, or the colorless precursors to anthocyanins 17. Leucoanthocyanidins are unstable and are quickly converted to anthocyanidins by anthocyanidin... 

FHT -flavanoneSjS-hydroxylase FLS -flavonol synthase DFR -dihydroflavonol reductase LAR - leucoanthocyanidin reductase 3GT - 3-Oglucosyltransferase... 

Dihydrocitral, 2891, 2892 Dihydrocorynantheol, 631 Dihydrodaidzein, 2444 Dihydrodiols, 1670 Dihydroergotamine, 707 Dihydroergotoxine, 707 Dihydroflavonol reductase, 1655 Dihydroflavonols, 1815... 

Scheme 50.1 Schematic of the major branch pathways of (poly)phenol biosynthesis. PAL phenylalanine ammonia-lyase, C4H cinnamate-4-hydroxylase, 4CL 4-coumaroyl CoA-ligase, CHS chalcone synthase, CHI chalcone isomerase, ANS anthocyanidin synthase, DPR dihydroflavonol reductase, FS flavone synthase, FLS flavonol synthase, F3H flavanone 3-hydroxylase...

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