Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)‐catechin to cyanidin and a novel procyanidin

Abstract

Anthocyanidins were proposed to derive from (+)-naringenin via (2R,3R)-dihydroflavonol(s) and (2R,3S,4S)-leucocyanidin(s) which are eventually oxidized by anthocyanidin synthase (ANS). Recently, the role of ANS has been put into question, because the recombinant enzyme from Arabidopsis exhibited primarily flavonol synthase (FLS) activity with negligible ANS activity. This and other studies led to the proposal that ANS as well as FLS may select for dihydroflavonoid substrates carrying a ''b-face'' C-3 hydroxyl group and initially form the 3-geminal diol by ''a-face'' hydroxylation. Assays with recombinant ANS from Gerbera hybrida fully supported the proposal and were extended to catechin and epicatechin isomers as potential substrates to delineate the enzyme specificity. Gerbera ANS converted (+)-catechin to two major and one minor product, whereas ent(À)-catechin (2S,3R-trans-catechin), (À)-epicatechin, ent(+)-epicatechin (2S,3S-cis-epicatechin) and (À)-gallocatechin were not accepted. The K m value for (+)-catechin was determined at 175 lM, and the products were identified by LC-MS n and NMR as the 4,4-dimer of oxidized (+)-catechin (93%), cyanidin (7%) and quercetin (trace). When these incubations were repeated in the presence of UDP-glucose:flavonoid 3-O-glucosyltransferase from Fragaria · ananassa (FaGT1), the product ratio shifted to cyanidin 3-O-glucoside (60%), cyanidin (14%) and dimeric oxidized (+)-catechin (26%) at an overall equivalent rate of conversion. The data appear to identify (+)-catechin as another substrate of ANS in vivo and shed new light on the mechanism of its catalysis. Moreover, the enzymatic dimerization of catechin monomers is reported for the first time suggesting a role for ANS beyond the oxidation of leucocyanidins.

The Grapevine R2R3-MYB Transcription Factor VvMYBF1 Regulates Flavonol Synthesis in Developing Grape Berries

“…5C). This is in line with previous results, suggesting an alternative route to produce flavonols in plants by takeover of DFR activity (Wilmouth et al, 2002;Wellmann et al, 2006;Stracke et al, 2009).…”

Nature’s assembly line: biosynthesis of simple phenylpropanoids and polyketides

“…Finally, leucoanthocyanidin reductase transforms this molecule into the final catechin (flavan-3-ol) product (Marles et al, 2003;Tanner et al, 2003;Wilmouth et al, 2002). Alternatively, anthocyanin synthase and anthocyanin reductase convert leucoanthocyanidins into ())-epicatechin, the epimer of (+)-catechin (Figure 6b) (Pang et al, 2007;Wellmann et al, 2006;Xie et al, 2003). Finally, regiospecific polymerization of leucoanthocyanidin, (+)-catechin, and ())-epicatechin yields proanthocyanidins.…”

Functional Analysis of a Predicted Flavonol Synthase Gene Family in Arabidopsis

“…Among these are flavonoid 3#-hydroxylase (F3#H) and flavonoid 3#,5#-hydroxylase, which mediate the addition of hydroxyl groups to the B ring of flavanones, flavones, dihydroflavonols, and flavonols (Hagmann et al, 1983;Kaltenbach et al, 1999), and dihydroflavonol reductase (DFR), which drives flux away from flavonols into anthocyanin and proanthocyanidin biosynthesis (Davies et al, 2003). More recently, anthocyanidin synthase (ANS) has been shown to use both dihydroflavonols and leucoanthocyanidins in vitro for the synthesis of flavonols, the latter suggesting an alternative route to quercetin using a substrate normally associated with anthocyanin and proanthocyanidin biosynthesis (Turnbull et al, 2004;Wellmann et al, 2006;Lillo et al, 2008). Some of the competition for common substrates appears to be mediated by the differential expression of genes required for upstream (flavonol) versus downstream (anthocyanin and proanthocyanidin) pathways (Pelletier et al, 1997;Mehrtens et al, 2005).…”

“…Yet, how these enzymes cooperate to control the metabolic balance among the branch pathways of flavonoid biosynthesis, possibly through participation in one or more enzyme complexes, remains to be fully determined. In fact, efforts to use enzymes such as FLS and ANS to engineer altered flavonoid profiles have had consistently unpredictable outcomes (Schijlen et al, 2006;Wellmann et al, 2006;Reddy et al, 2007).…”