Flavanone 3 3-Hydroxylase

Figure 6.1 Major branch pathways of flavonoid biosynthesis in Arabidopsis. Branch pathways, enzymes, and end products present in other plants but not Arabidopsis are shown in light gray. Abbreviations cinnamate-4-hydroxylase (C4H), chalcone isomerase (CHI), chalcone synthase (CHS), 4-coumarate CoA-ligase (4CL), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), flavonoid 3 or 3 5 hydroxylase (F3 H, F3 5 H), leucoanthocyanidin dioxygenase (LDOX), leucoanthocyanidin reductase (LCR), O-methyltransferase (OMT), phenylalanine ammonia-lyase (PAL), rhamnosyl transferase (RT), and UDP flavonoid glucosyl transferase (UFGT).

PELLETIER, M.K., SHIRLEY, B.W., Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings Coordinate regulation with chalcone synthase and chalcone isomerase, Plant Physiol., 1996, 111, 339-345. 

CL, 4-coumarate CoA ligase CHS, chalcone synthase CHI, chalcone isomerase F3H, flavanone 3-hydroxylase DFR, dihydroflavonol 4-reductase ANS, anthocyanidin synthase FGT, flavonoid 3-O-glucosyltransferase. 

Flavanone 3[)-hydroxylase (F3H, FHT EC 1.14.11.9) is a selective enzyme that catalyzes the stereospeciflc 3[)-hydroxylation of 2S-flavanones to form 2R,3R... 

Flavanone 3 -hydroxylase (F3 H ECl.14.13.21 CYP75B) activity was initially identified in microsomal preparations of golden weed (Haplopappus gracilis) [110]. E3 H from irradiated parsley cell cultures was later biochemically analyzed and characterized as a cytochrome P450 having an absolute requirement for NADPH and molecular oxygen as cofactors [111]. The enzyme has been shown to have activity with flavanones, flavones, dihydroflavonols, and flavonols, but does not appear to have activity with anthocyanidins [111]. The first cDNA clone for E3 H was isolated from Petunia [112]. It has been suggested that E3 H may serve as an anchor for the proposed flavonoid multi-enzyme complex on the cytosolic surface of the endoplasmic reticulum [44]. 

Britsch L, Grisebach H (1986) Purification and characterization of (2S)-flavanone 3-hydroxylase from Petunia hybrida. Eur J Biochem 156(3) 569-577... 

Lukacin R, Britsch RLE (1997) Identification of strictly conserved histidine and arginine residues as part of the active site in Petunia hybrida flavanone 3 )-hydroxylase. Eur J Biochem 249 748-757... 

Lukacin R, Groning 1, Scltiltz E, Britsch L, Matem U (2000) Purification of recombinant flavanone 3 )-hydroxylase from Petunia hybrida and assignment of the primary site of proteolytic degradation. Arch Biochem Biophys 375(2) 364—370... 

Wellmann F, Matern U, Lukacin R (2004) Significance of C-terminal sequence elements for petunia flavanone 3 )-hydroxylase activity. FEBS Lett 561(1-3) 149-154... 

Owens DK, Crosby KC, Runac J, Howard B, Winkel BS (2008) Biochemical and genetic characterization of arabidopsis flavanone 3-hydroxylase. Plant Physiol Biochem 46 833-843... 

Halbwirth H, Fischer TC, Schlangen K, Rademacher W, Schleifer K, Forkmann G, Stitch K (2006) Screening for inhibitors of 2-oxoglutarate-dependent dioxygenases flavanone 3-hydroxylase and flavonol synthase. Plant Sd 171 194-205... 

Pelt JL, Downes WA, Schoborg RV, McIntosh CA (2003) Flavanone 3-hydroxylase expression in Citrus paradisi and Petunia hybrida seedlings. Phytochemistry 64(2) 435-444... 

Deboo, G.B., Albertsen, M.C., and Taylor, L.P., Flavanone 3-hydroxylase transcripts and flavo-nol accumulation are temporally coordinate in maize anthers. Plant J., 7, 703, 1995. 

Zuker, A. et al.. Modification of flower colour and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Mol Breed., 9, 33, 2002. 

Increases in substrate levels that were accidentally produced by metabolic engineering also resulted in an olfaction-detectable increase in the methyl benzoate emission in transgenic carnation [30]. The metabolic flux from the anthocy-anin pathway was redirected towards benzoic acid, the methyl ester precursor, by antisense suppression of the flavanone 3-hydroxylase. 

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.

Figure 3-7. Flavonoid biosynthesis (this page and next page). The enzymes involved in this pathway are (a) chalcone synthase (E.C. 2.3.1.73), (b) aureusidin synthase (E.C. 1.21.3.6), (c) chalcone isomerase (E.C. 5.5.1.6), (d) flavanone 3-hydroxylase (E.C. 1.14.11.9), (e) flavone synthase (E.C. 1.14.11.22), (f) flavonoid 3 -hydroxylase (E.C. 1.14.13.21),...

Figure 1.36 Schematic diagram of the stilbene and flavonoid biosynthetic pathway. Enzyme abbreviations SS, stilbene synthase CHS, chalcone synthase CHR, chalcone reductase CHI, chalcone isomerase IFS, isoflavone synthase FNS, flavone synthase F3H, flavanone 3-hydroxylase FLS, flavonol synthase F3 H, flavonoid 3 -hydroxylase DFR, dihydroflavonol 4-reductase LAR, leucoanthocyanidin 4-reductase LDOX, leucocyanidin deoxygenase ANR, anthocyanidin reductase EU, extension units TU, terminal unit.

In plants accumulating anthocyanins, flavonols, and proanthocyanidins, naringenin is stereospecifically hydroxylated at position 3 of the C-ring (C3) by the 2-oxoglutarate-dependent dioxygenase flavanone 3-hydroxylase (F3H, EC 1.14.11.9) to yield the 3-hydroxy-trans-flavanone (syn. dihydroflavonol) dihy-drokaempferol [Springob et al., 2003] (Fig.21 2). Dihydroquercetin (3, 4, 5,5, 7-... 

Comparison of the predicted amino acid sequence of the protein encoded by pTOM 13 with the sequence of flavanone 3-hydroxylase showed considerable similarity (A. Prescott and C. Martin, personal communication, cited in Hamilton etal., 1990). This is particularly interesting, since it has been suggested that the conversion of ACC to ethylene might involve a hydroxylation reaction (Yang, 1985). Armed with this information and using conditions developed for extraction and purification of flavanone 3-hydroxylase, Ververidis John (1991) showed that it is possible to solubilise the ethylene forming enzyme from plants and retain full... 

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.

FORKMANN, G., HELLER, W., GRISEBACH, H Anthocyanin biosynthesis in flowers of Matthiola incana flavanone 3-hydroxylase and flavonoid 3 -hydroxylase. Z. Naturforsch, 1980,35c, 691-695. 

FORKMANN, G., STOTZ, G., Selection and characterization of flavanone 3-hydroxylase mutants of Dahlia, Streptocarpus, Verbena and Zinnia. Planta, 1984, 161,261-265. 

CHARRIER, B., LEROUX, C., KONDOROSI, A., RATET, P., The expression of alfalfa flavanone 3-hydroxylase promoter-gus fusion in Nicotiana benthamiana correlates with the presence of flavonoids detected in situ. Plant Mol. Biol., 1996, 30, 1153-1168. 

Flavanone 3-hydroxylase (F3H) converted naringenin (1) to dihydrokaem-pferol (3-OH-flavanone) (31). Flavonoid 3 -hydroxylase (F3 II) and flavonoid 3 5 -hydroxylasc (r3 5 I I) converted dihydrokaempferol (31) to dihydroflavo-nols (3-OH-flavanones) (32) such as dihydro quercetin (33) and dihydromy-ricetin (34). 

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