Anthocyanidin synthase

The role of anthocyanidin synthase (ANS) in the biosynthetic pathway is to catalyze reduction of the leucoanthocyanidins to the corresponding anthocyanidins. However, in vivo it is anthocyanidins in pseudobase form that are formed, as is described below. In this chapter, use of anthocyanidin should be taken to include the pseudobase form. Furthermore, although the name ANS is commonly used, the enzyme is also referred to in the literature as leucoantho-cyanidin dioxygenase (LDOX), reflecting the reaction type. 

Much of the information on ANS has come not from studies on enzyme extracts but from analysis of DNA sequences and recombinant proteins. Sequences for the ANS were first isolated using transposon generated mutant lines of A. majus and Z. mays They encoded proteins of 40 to 41 kDa that were found to have similarity to 20GDs, during a study on a nonflavonoid enzyme. This sequence-based identification was confirmed by the in vitro assay of the recombinant Perilla frutescens protein, and subsequent assays on recombinant ANS from a range of species that confirmed the requirement for Fe, 20G, and ascorbate. Sequence comparisons show that ANS is more closely related to flavonol synthase (FLS), another 20GD, than to F3H. 

Some of the 20GDs of flavonoid biosynthesis (F3H, FNSI, FLS, and ANS) have been studied for acceptance of (9-methylated substrates. Substrates methylated at the 3 -hydroxyl were accepted, while methylation at the 4 - or 7-hydroxyls reduced activities to varying degrees. Multiple methylation or methylation at other positions prevented acceptance of the substrate. 

For 3-deoxyanthocyanin biosynthesis the 3-hydroxyl is, of course, lacking from the ANS substrates (e.g., apiforol). Whether a specific ANS is thus involved in 3-deoxyanthocyanin biosynthesis is not clear. However, it has been postulated that the reaction may still proceed through 3-hydroxylation, and initial results suggest recombinant ANS from species that do not produce 3-deoxyanthocyanins may still use apiforol as a substrate to produce apigenini-din (results of J-I. Nakajima and K. Saito of Chiba University, Japan, with the authors coworkers in New Zealand). 

The F3GT is part of the UDPG-glycosyltransferase (UGT) family, which is family 1 of the glycosyltransferase superfamily (EC 2.4.l.X). UGTs have a central role in detoxifying or 

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Shimada, S., Inoue, Y., and Sakuta, M., Anthocyanidin synthase in non-anthocyanin-producing Caryophyllales species. Plant J., 44, 950, 2005. 

Figure 5.4. Abbreviated scheme for biosynthesis of major flavonoid subclasses, showing the primary enzymes and substrates leading to different subclasses. Bold-faced, uppercase abbreviations refer to enzyme names, whereas substrate names are presented in lowercase letters. PAL, phenylalanine ammonia lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA ligase CHS, chalcone synthase CHI, chalcone isomerase CHR, chalcone reductase IPS, isoflavone synthase F3H, flavonone 3-hydroxylase F3 H, flavonoid 3 -hydroxylase F3 5 H, flavonoid 3 5 -hydroxylase FNSI/II, flavone synthase DFR, dihydroflavonol 4-reductase FLS, flavonol synthase ANS, anthocyanidin synthase LAR, leucoanthocyanidin reductase ANR, anthocyanidin reductase UFGT, UDP-glucose flavonoid 3-O-glucosyltransferase. R3 = H or OH. R5 = H or OH. Glc = glucose. Please refer to text for more information.

Turnbull JJ, Nakajima J, Welford RW, Yamazaki M, Saito K and Schofield CJ. 2004. Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis anthocyanidin synthase, flavonol synthase, and flavanone 33-hydroxylase. J Biol Chem 279 1206-1216. 

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. 

Wilmouth RC, TumbuU JJ, Welford RW, Clifton U, Prescott AG, Schofield CJ (2002) Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana. Structure 10(1) 93-103... 

Saito K, Kobayashi M, Gong Z, Tanaka Y, Yamazaki M (1999) Direct evidence for anthocyanidin synthase as a 2-oxoglutarate-dependent oxygenase molecular cloning and functional expression of cDNA from a red form of Perilla frutescens. Plant J 17(2) 181-189... 

Turnbull JJ, Sobey WJ, Aplin RT, Hassan A, Firmin JL, Schofield CJ, Prescott AG (2000) Are anthocyanidins the immediate products of anthocyanidin synthase Chem Commun 24 2473-2474... 

ANTHOCYANIDIN SYNTHASE ANTHRANILATE 1,2-DIOXYGENASE ANTHRANILATE HYDROXYLASE... 

Turnbull, J.J. et al.. Purification, crystallization and preliminary x-ray diffraction of anthocyanidin synthase from Arabidopsis thaliana. Acta Crystallogr. D, 57, 425, 2001. 

Turnbull, J.J. et al.. The C-4 stereochemistry of leucocyanidin substrates for anthocyanidin synthase affects product selectivity. Bioorg. Med. Chem. Lett., 13, 3853, 2003. 

Rosati, C. et al., Engineering of flower colour in forsythia by expression of two independently-transformed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway. Mol Breed, 12, 197, 2003. 

Anthocyanidin synthase (ANS) is the enzyme that dehydrates the leucoanthocyanidins (Figure 3-8). While this enzyme had been postulated to exist, and cDNA s encoding the putative ANS had been identified (e.g. A2 in... 

Figure 3-8. Biosynthesis of anthocyanins and condensed tannins. The enzymes involved in this pathway are (a) anthocyanidin synthase (E.C. 1.14.11.19), (b) anthocyanin 3-glycosyl transferase, and (c) BANYULS.

The branch pathway for anthocyanin biosynthesis starts with the enzymatic reduction of dihydrofiavonols to their corresponding flavan 3,4-diols (leucoanthocyanidins) by substrate-specific dihydroflavonol 4-reductases (DFR). Flavan 3,4-diols are the immediate precursors for the synthesis of catechins and proanthocyanidins. Catechins are formed by enzymatic reduction of the flavan 3,4-diols in the presence of NADPH to leucoanthocyanidins, which are subsequently converted to anthocyanidins by the 2-oxoglutarate-dependant dioxygenase, anthocyanidin synthase. Further glycosylation, methylation, and/or acylation of the latter lead to the formation of the more stable, colored anthocyanins (Scheme 1.1). The details of the individual steps involved in flavonoid and isoflavonoid biosynthesis, including the biochemistry and molecular biology of the enzymes involved, have recently appeared in two excellent reviews.7,8... 

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.

The flavonoid hydroxylases (F3 H, F3 ,5 H) and the phenylpropanoid enzyme cinnamate 4-hydroxylase (C4H) are cytochrome P-450s that are anchored to the cytoplasmic face of the ER.14 F3 H and F3 ,5 H add hydroxyl groups to the 3 and/or 5 of the B ring bringing about chemical and spectral diversity to the flavonoids. Dihydroflavonol 4-reductase (DFR) is the committed step to the production of the precursor of the colored compounds - the leucoanthocyanidins (Fig. 3.2). It is upon the reduction of the 4 keto of the C -ring by DFR (Fig. 3.1) and its further reduction by leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/AS) that the de-localization of the electrons necessary for the formation of the planar flavylium ion is permitted.2... 

Leucoanthocyanidin dioxygenase (LDOX), an anthocyanidin synthase (ANS), converted luecoanthocyanidins (flavan-3,4-diols, 37) to 3-hydroxy-anthocyanidins (38). The 3-hydroxy-anthocyanidins (38) were converted to epi-flavan-3-ols (52) by anthocyanidin reductase (ANR). Then, epz-flavan-3-ols (52) were converted to brownish condensed tannins (proanthocyanidins, PA, 44) by condensing enzyme (CE). Similarly, condensed tannins (proanthocyanidins, PA, 44) were converted to oxidized tannins (oxidized proanthocyanidins, 25) by proanthocyanidine oxidase (PRO) (Fig. 10) [23,24],... 

The proanthocyanidins share a biosynthesis sequence with the anthocyanins, a class of flavanoids well understood in biochemical and genetic terms. Three enzymes, leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS), and anthocyanidin reductase (ANR), function at branches between the anthocyanin and proanthocyanidin biosynthesis pathways (Scheme 1). LAR is a member of the reductase—eptmerase—dehydrogenase enzyme... 

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.

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