A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity

Abstract

BackgroundDihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood.ResultsBy using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds.ConclusionsWe isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.Electronic supplementary materialThe online version of this article (10.1186/s12870-017-1200-6) contains supplementary material, which is available to authorized users.

Computational modeling of anthocyanin pathway evolution

“…For example, DFR ( Fig. 1) can experience a mutation that improves its activity on one substrate without changing activity on other substrates, consistent with the dierent K M and K cat values for dierent substrates that have been measured experimentally (Fischer et al 2003;Katsu et al 2017;Miyagawa et al 2015). While it might be biologically reasonable to assume some cost of improvement on one substrate for activity on other substrates (Kaltenbach and Tokuriki 2014;…”

Biosynthesis and regulation of flavonoids in buckwheat

“…Buckwheat contains only cyanidin, in the forms of cyanidin-3rutinoside (cyanidin-3-glucoside-rhamnoside), cyanidin-3- glucoside, cyanidin-3-galactoside, and cyanidin 3galactopyranosyl-rhamnoside (Kim et al 2007, Watanabe 2007. This specificity is related to the substrate preference of dihydroflavonol 4-reductase (DFR) of buckwheat (see 3-2.9; Katsu et al 2017).…”

“…Anthocyanins in buckwheat accumulate mainly at the base of the stem, which fades in color toward the top, and their composition differs depending on the location in the stem (Eguchi et al 2008). Some anthocyanins may be transported from roots or the base of the buckwheat stem to the top, since genes involved in anthocyanin synthesis are expressed in roots (Katsu et al 2017), but further study is needed to clarify this.…”

Characterization of novel mutants of hexaploid wheat (Triticum aestivum L.) with various depths of purple grain color and antioxidant capacity

“…DFR is a key enzyme in the anthocyanin biosynthesis pathway that catalyzes the reduction of dihydroflavols such as dihydrokaempferol, dihydroquercetin and dihydromyricetin to lecoanthocyanins (pelargonidin, cyanidin and delphinidin). 31 DFR activity is associated with anthocyanin content in various plants with different colors, 32,33 suggesting that DFR plays a significant role in the expression of anthocyanin biosynthesis genes in developing colored wheat grains. The anthocyanin biosynthesis pathway is regulated not only by anthocyanin structural genes but also by the MYB-bHLH-WD40 (MBW) complex.…”