Leucoanthocyanidin reductase

LAR removes the 4-hydroxyl from leucoanthocyanidins to produce the corresponding 2,3-tran5-flavan-3-ols, e.g., catechin from leucocyanidin. Despite early biochemical characterization, it is only recently that a LAR cDNA was isolated and the encoded activity characterized in detail. Tanner et al. purified LAR to homogeneity from Desmodium uncinatum (silverleaf desmodium), and used a partial amino acid sequence to isolate a LAR cDNA. The cDNA was expressed in E. coli, N. tabacum, and Trifolium repens (white clover), with the transgenic plants showing significantly higher levels of LAR activity than nontransformed plants. 

---

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. 

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).

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.

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. ... 

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. 

Tanner GJ, Franki KT, Abrahams S, Watson JM, Larkin PJ, Ashton AR. 2003. Proanthocyanidin biosynthesis in plants Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA. J Biol Chem 278 31647-31656. 

Leucoanthocyanidin reductase (LAR) converted leucoanthocyanidins (flavan-3,4-diols, 37) to flavan-3-ols (40). The flavan-3-ols (40) were converted to condensed tannins (proanthocyanidins, PA, 44) by condensing enzyme (CE). Following this condensed tannins (proanthocyanidins, PA, 44) finally yielded their oxidized tannins (oxidized proanthocyanins, 45) by proanthocyanidine oxidase (PRO) (Fig. 9) [23,24]. 

The biosynthetic pathways of epicatechin (55), cyanidin (51) and proan-thocyanidin (PA) (44) had the same intermediates to leucocyanidin (48). Next, leucoanthocyanidin reductase (LAR) converts the leucocyanidin (48) to epicathechin (55), whereas leucoanthocyanidin dioxygenase (LDOX) converted leucocyanidin (48) to cyanidin (51). Two cyanidin (51) and epicathechin (55) are a precursor of proanthocyanidins (PA) (44). 

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. 

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

Leucoanthocyanidin reductase (EAR), which is related to the isoflavoue reductase group of plaut enzymes, catalyzes the reduction of leu-coanthocyanidins to (+)-afzelechin, (+)-cate-chin, and (+)-gallocatechin, building blocks for PA biosynthesis [77] (Fig. 5). The above cate-chin series of flavan-3-ols possess 2,3-trans stereochemistry. The corresponding (ep/)-cate-chin series with 2,3-cis stereochemistry is formed by a different mechanism involving an unrelated reductase (ANR) that acts at the level of anthocyanidin [90] (Fig. 5). 

It is still not known whether the condensation of monomers proceeds by enzymatic or non-enzymatic mechanisms, or by both mechanisms. The main building units are (ra s-2,3-flavan-3-ols, that is (2i ,3S)-isomers and cis-2,3-flavan-3-ols that are (2J ,3J )-isomers. The majority of proanthocyanidins arise from (+)-catechin, which is the lower unit and (-)-epicatechin, which is the upper unit in proanthocyanidins. It is assumed that the formation of the upper units involves quinones or carbocations arising from leucoanthocyanidins, anthocyanidins and flavan-3-ols and perhaps some enzymes, such as leucoanthocyanidin reductases and polyphenol oxidases. The expected mechanism of formation of these proanthocyanidin units is outlined in Figure 8.95. 

---