DFR substrate specificity - completing the puzzle

Dihydroflavonol 4-reductase (DFR) is a key enzyme in flavonoid biosynthesis in plants and catalyzes the formation of anthocyanin precursors. The natural substrates for the DFR are dihydrokaempferol, dihydroquercetin and dihydromyricetin, which differ in their chemical structure by a different number of hydroxyl groups (OH groups) in the so-called B ring. The substrate specificity of DFRs can vary, with some DFRs accepting all three substrates equally, while others have high specificity for particular substrates. Since the number of OH groups in the substrate determines the color of the anthocyanins, the substrate specificity of the DFR is a decisive factor in the different expression of flower or fruit colors. The substrate specificity is determined by slight differences in the amino acid sequence in the region of the substrate binding site and thus slightly altered structure of the enzyme. Although numerous studies are available on the substrate specificity of the DFR, there is still no systematic understanding of how precisely this specificity is determined at the molecular level of the amino acid sequence. The aim is to establish the structure-function relationship of the DFR in terms of its specificity to the three substrates. In the project, various DFRs from plants are produced in bacterial cultures and their substrate specificity is examined using enzyme assays. The results are related to the respective amino acid sequences in the region of the substrate binding site. In particular, the DFR from the grapevine is of importance, since the crystal structure of the enzyme is known. This enables the complementary combination of "in silico" techniques (theoretical enzyme modeling using special software) with experimental data from the enzyme assays. In this way, the effects of changes in the amino acid sequence can be predicted and verified experimentally, and vice versa the corresponding substrate specificity can be derived in the model based on the amino acid sequences. This is supported by the generation of point mutations in the enzyme, where one or more amino acids in the region of the substrate binding site are specifically exchanged and the effect on the substrate specificity is determined. Ultimately, it should be possible to predict substrate specificity based on amino acid sequence information. The precise understanding of the substrate specificity at the level of the amino acid sequence contributes significantly to the targeted breeding of plants with new flower and fruit colors. Furthermore, the project deals with the evaluation of different DFR enzyme tests to enable robust and comparable results within the research community.