Anthocyanidin reductase

Like LAR, ANR is a member of the RED protein family. Full details of the protein structure and reaction mechanism are yet to be published. However, Xie et al. compared the A. thaliana (AtANR) and M. truncatula (MtANR) ANR amino acid sequences and recombinant protein activities, and made suggestions on the possible reaction series. The two recombinant proteins showed significantly different kinetic properties, substrate specificities, and cofactor requirements. Although AtANR and MtANR share only 60% sequence identity, some well-conserved domains are evident, in particular the Rossmann dinucleotidebinding domain (GxxGxxG) near the N-termini. However, two amino acid variations did 

The ANR reaction involves a double reduction at the C-2 and C-3 of the anthocyanidin, allowing the inversion of C-3 stereochemistry. Xie et al. postulate four possible reaction mechanisms, proceeding via either flav-3-en-ol or flav-2-en-ol intermediates. The proposed reaction mechanisms are based on anthocyanidins (the flavylium cation forms) as the starting molecules however, as the authors acknowledge, other forms of the anthocyanidin may exist in vivo. In particular, the 3-flaven-2,3-diol pseudobase is thought to be the more likely in vivo product of the ANS. 

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Fig. 5 Scheme of the flavonoid pathway leading to synthesis of proanthocyanidins. The enzymes involved in the pathway are shown as follows CHS = chalcone synthase CHI = chalcone isomerase F3H = flavanone-3B-hydroxylase DFR = dihydroflavonol-4-reductase LDOX = leucoanthocynidin dioxygenase LAR = leucoanthocyanidin reductase ANR = anthocyanidin reductase adapted from [27] and [28]... 

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.

Flavanols and procyanidins Flavanols, or flavan-3-ols, are synthesized via two routes, with (+) catechins formed from flavan-3,4-diols via leucoanthocyanidin reductase (LAR), and (—) epicatechins from anthocyanidins via anthocyanidin reductase (ANR) (see Fig. 5.4). These flavan-3-ol molecules are then polymerized to condensed tannins (proanthocyanidins or procyanidins), widely varying in the number and nature of their component monomers and linkages (Aron and Kennedy 2008 Deluc and others 2008). It is still not known whether these polymerization reactions happen spontaneously, are enzyme catalyzed, or result from a mixture of both. 

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. ... 

Xie, D.-Y. et al., Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid... 

Xie, D.-Y., Sharma, S.B., and Dixon, R.A., Anthocyanidin reductases from Medicago truncatula and Arabidopsis thaliana. Arch. Biochem. Biophys., 422, 91, 2004. 

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.

Bogs J, Downey MO, Harvey JS, Ashton AR, Tanner GJ, Robinson SP. 2005 Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol 139 652-663. 

Paolocci F, Robbins MP, Madeo L, Arcioni S, Martens S, Damiani F. 2007. Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. Structure, expression, analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus. Plant Physiol 143 504-516. 

Xie DY, Sharma SB, Wright E, Wang ZY, Dixon RA. 2006. Metabolic engineering of proanthocyanidins through co-expression of anthocyanidin reductase and the PAP1 MYB transcription factor. Plant J 45 895-907. 

Pfeiffer J, Kiihnel C, Brandt J, Duy D, Punyasiri PA, Forkmann G, Fischer TC. 2006. Biosynthesis of flavan 3-ols by leucoanthocyanidin 4-reductases and anthocyanidin reductases in leaves of grape (Vitis vinifera L.), apple (Malus x domestica Borkh.) and other crops. Plant Physiol Biochem 44 323-334. 

Shen G-A, Pang Y, Wu W, Liu X, Zhao L, Sun X, Tang KJ. 2005. Isolation and characterization of putative anthocyanidin reductase gene from Ginkgo biloba. Plant Physiol 163 224—227. 

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 anthocyanidin reductase enzyme recently described in Arabidopsis and Medicago was shown to be present in tea with very high activity and could produce epicatechin as well as epigallocatechin from the respective anthocyanidins, thus explaining very high contents of llavan-3-ols. Especially, two enzymes, dihydroflavonol 4-reductase and leucoanthocyanidin 4-reductase selectively catalyze key steps in their biosynthesis of catechins (43,62) and gallocatechins (63,64), respectively (Fig. 14) [31]. 

Then, BANYULS (BAN) genes from Arabidopsis thaliana and Medicago truncatula encode anthocyanidin reductase, which could convert anthocyani-dins to the corresponding 2,3-czs-flavan-3-ols (43, 62, 63, 64). Interestingly, the ectopic expression of BAN in tobacco flower petals and Arabidopsis leaves induced the accumulation of condensed tannins without production of 2,3-ds-flavan-3-ols (43,62,63, 64) (Fig. 14) [32],... 

By reduction of dihydroquercetin and dihydromyricetin, operated by the enzyme dihydroflavonol-4-reductase (DFR) (in vine the reduction of dihydrokaempferol does not occur), 2,3-trans leucocyanidin and leucodelphinidin form. Successive synthesis of (+)-catechin and (+)-gallocatechin (2,3-trans isomers) is mediated by the enzyme leucoan-thocyanidin reductase (LAR). On the other hand, the synthesis of (—)-epicatechin and (—)-epigallocatechin (2,3-cis isomers) proceeds via oxidation of 2,3-trans leucoanthocyanins into cyanidin and delphinidin (accompanied with chirality loss) mediated by the enzyme leucoan-thocyanidin dioxygenase (LDOX), which is followed by the reduction catalyzed by the enzyme anthocyanidin reductase (ANR) with reacquisition of chrality (Fujita et al., 2005). 

Fujita, A., Soma, N., Goto-Yamamoto, N., Shindo, H., Kakuta, T., Koizumi, T. and Hashizume, K. (2005) Anthocyanidin reductase gene expression and accumulation of flavan-3-ols in grape berry, Am. J. Enol. Vitic., 56(4), 336-342. 

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.

It has been experimentally demonstrated that all anthocyanin pigments are derived from one of three aglycones (pelargonidin, cyanidin and delphinidin). The differences in color of anthoeyanins are due to differences in the hydroxylation and methylation pattern as well as the number and type of sugars. Leucocyanidin can be enzymatically converted to catechin (leucoanthocyanidin reductase). Likewise, cyanidin can be enzymatically converted to epicatechin (anthocyanidin reductase). These two compoimds are the building blocks of the oligo-Zpolymeric proanthocyanidins. 

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