Molecular background of the flavanone 4-reductase activity

Dihydroflavonol 4-reductase (DFR) is a key enzyme of the flavonoid pathway catalyzing the formation of precursors for the biosynthesis of anthocyanin pigments and monomeric flavan 3-ols, the building blocks of tannins. Although dihydroflavonols are the main substrates, flavanones can be converted as well. The latter reaction is known as flavanone 4-reductase (FNR) activity. The two flavonoid classes differ by a hydroxyl group in position 3 of the heterocyclic ring. This project will study the ability of the enzyme DFR to accept flavanones beside dihydroflavonols as substrates on the molecular level. The majority of DFRs show a strong dihydroflavonol preference but we identified two further DFR types which either accept exclusively dihydroflavonols or both substrates to a similar extent. Theproject will characterizethethree DFR typeswith differing flavanone/dihydroflavonol acceptance and identify further representatives of the so far rare types. Sequence comparison of the three DFR types revealed five significant differences in the amino acid sequence around the presumed substrate recognition sites which could be essential for the coordination of the hydroxyl group in position 3 and therefore be decisive for the ability/inability to convert dihydroflavonols and flavanones, respectively. Kinetic studies will be performed with GST-tag purified recombinant DFRs from different plant species using dihydroflavonols and flavanones as substrates in the assays. Results will be correlated with information of amino acid sequence at those positions, which are assumed to be responsible for flavanone/dihydroflavonol acceptance. In a next step, site specific mutations will be performed to alter substrate acceptance according to our hypotheses. Finally, amino acid residues and positions which determinate the acceptance of dihydroflavonols and/or flavanones will be identified. In-depth understanding of the first committed step in the formation of 3-deoxyflavonoids will be an important contribution for the elucidation of the biosynthesis of 3-deoxyanthocyanidin phytoalexins and antimicrobial compounds in general and could help to understand host- pathogen interactions.

Dihydroflavonol 4-reductase (DFR) is a key enzyme of the flavonoid pathway catalyzing the formation of flavan 3,4-diols. Although dihydroflavonols are the main substrates, flavanones can be converted as well (flavanone 4-reductase (FNR) activity). Substrate specificity of DFR is complex and has great influence on the flavonoid spectrum formed in a plant. The project focused on DFR at the molecular level by exploiting DFR types showing contrasting substrate specificity (i) regarding dihydroflavonol/flavanone acceptance in general, and (ii) regarding the hydroxylation pattern in the B-ring for maize DFRs A1 and A1*. In silico analysis of the presumed substrate recognition site of DFRs together with different crystal structure models enabled us to select specific amino acids, which could be responsible for the determination of the respective substrate specificity. We cloned several DFRs from various plant species, heterologously expressed them in E. coli and tested their substrate specificity with enzyme assays. Site-directed mutagenesis and the creation of chimeric DFRs were performed to evaluate the possibility for an interconversion between different DFR types. Concerning dihydroflavonol/flavanone acceptance, a clear bidirectional interconversion between the DFR types remains elusive, however, some tendencies became evident, and further steps in understanding possible interactions at the presumed substrate binding site could be carried out. Kinetic data for maize A1 and A1* DFRs revealed a contrasting substrate specicity regarding the B-ring hydroxylation pattern. By use of site-directed mutagenesis we defined amino acids that are responsible for the divergent substrate specificities. The complete interconversion between these two DFRs could be achieved by altering only 2-3 amino acids. Moreover, an event-specific qualitative PCR method for the detection of a specific genetically modified orange petunia harbouring the maize A1 DFR from a 1980s scientic transgenic breeding attempt was developed. Results from the project will help to better understand interactions of the substrate/protein complex of DFRs and open new possibilities for the creation of plants with a modified flavonoid spectrum relevant for e.g. flower colour, dietary composition or phytopathological matters.