Dihydroflavonol 4-Reductase (DFR) substrate specificity - Completing the puzzle. – DFR-SPEC

Dihydroflavonol 4-reductase (DFR) substrate specificity – Completing the puzzle

Wider research context
Dihydroflavonol 4-reductase (DFR) is a key enzyme in the biosynthesis of anthocyanins and catalyzes the reduction of dihydroflavonols to leucoanthocyanidins. Some DFRs can convert dihydrokaempferol (DHK), dihydroquercetin (DHQ) and dihydromyricetin (DHM) irrespective of their B-ring hydroxylation pattern, whereas others show distinct substrate specificity. Differences apparently come from subtle mutations of the amino acid sequence. Despite an abundance of studies on DFR substrate specificity, there is still no systematic understanding of how the specificity is determined at the molecular level. This project proposal addresses the DFR substrate specificity and structure-function relationship with respect to the B-ring hydroxylation pattern of the substrates.

Hypotheses
H1: Substrate specificities found in literature are frequently tainted by biases inherent in the test systems and, therefore, not necessarily comparable. This hinders reliable conclusions about the structure-function relationships. Evaluation of the common assay systems is imperative for the scientific community.
H2: At least four types of DFRs should be distinguished (unspecific, DHK preferring, DHM preferring, DHK averse). So far, no consistent explanation for the substrate specificity on the molecular level is available.
H3: After generating and consolidating data for DFR substrate specificity, it is possible to predict substrate specificity from the amino acid sequence and to point at the atomic level the discriminant points at the basis of these differences.
H4: Considering the low expression rates of the maize DFR in the transgenic Petunia, we hypothesize that the DHM preferring DFR of Petunia is able to convert DHK under exceptional conditions.

Approach
We will analyze the most commonly used DFR assay types to understand the reasons for inconsistent results. Availability of the crystal structure of the Vitis DFR allows the combination of experimental results from enzyme assays (including site-directed mutations of the Vitis DFRs) and theoretical molecular modelling (including substrate lability in the enzyme cavity) in silico and vice versa. Based on the results a schematic to predict DFR substrate specificity from the amino acid sequences will be established.

Level of originality
The unique combination of complementary expertise of the project partners will enable the comprehensive definition, molecular description and prediction of DFR substrate specificity at the level of sequence information. Moreover, evaluated DFR assay(s) will be provided to allow the generation of comparable enzyme results.

Primary researchers involved
Dr. Christian Haselmair-Gosch, Technische Universität Wien, Institute of Chemical, Environmental and Bioscience Engineering, ORCID 0000-0003-3231-0962
Prof. Serge Antonczak, Université Côte d'Azur, Institut de Chimie de Nice, ORCID 0000-0003-2758-8409