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

BMC Plant Biology

Research Article

Amanda R. Walker¹  Yasuo Yasui²  Seiji Kubota³  Motoyuki Koshio³  Toshiyuki Komori³  Kiyoshi Furukawa³  Toshimasa Yamazaki⁴  Wataru Tsuchiya⁴  Noritoshi Inagaki⁴  Rintaro Suzuki⁴  Kenjiro Katsu⁵  Tomomi Hisano⁵  Katsuhiro Matsui⁶ 

[1] CSIRO Agriculture & Food, Wine Innovation West, Hartley Grove, 5064, Urrbrae, SA, Australia;Graduate School of Agriculture, Kyoto University, Yoshida-honmachi, Sakyou-ku, 606-8501, Kyoto, Japan;Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University, Kamitomioka 1603-1, Nagaoka, 940-2188, Niigata, Japan;NARO, Advanced Analysis Center, Kannondai 2-1-2, Tsukuba, 305-8602, Ibaraki, Japan;National Agriculture and Food Research Organization (NARO), Kyushu Okinawa Agricultural Research Center, Suya 2421, Koshi, 861-1192, Kumamoto, Japan;National Agriculture and Food Research Organization (NARO), Kyushu Okinawa Agricultural Research Center, Suya 2421, Koshi, 861-1192, Kumamoto, Japan;Present address: NARO, Institute of Crop Science, Kannondai 2-1-2, Tsukuba, 305-8518, Ibaraki, Japan;

关键词: 3D structure modelling;    Anthocyanins;    Fagopyrum esculentum;    Flavonoid;    Recombinant protein;    Substrate preference;   

DOI  :  10.1186/s12870-017-1200-6

 received in 2017-03-16, accepted in 2017-12-01,  发布年份 2017

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© The Author(s). 2017