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.
Highlights
Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway
Production of recombinant DFR proteins in Escherichia coli To determine whether the buckwheat DFR proteins that we identified can catalyse dihydroflavonols and show substrate specificity, we produced N-terminal His-tagged recombinant DFR proteins based on the methods of Shimada et al [9]
Each band encoded a peptide with high sequence similarity to DFRs of other plant species, and we initially designated them as FeDFR1 and FeDFR2
Summary
Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. 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. DFR proteins in many plants mainly catalyse the reduction of three different substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin) into leucopelargonidin, leucocyanidin, and leucodelphinidin, respectively (Fig. 1). Johnson et al [7] used molecular biological methods, such as the production of transgenic plants with chimeric DFRs, to identify a region of DFR proteins that consists of 26 amino acids and that determines substrate specificity. They found an important residue that determines the preference against the three substrates in the region that determines substrate specificity. The molecular basis of the substrate preference of DFR is not yet fully understood
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