Tutkimusmatka dihydroflavonoli 4-reduktaasin substraatin tunnistukseen

Plants are vital to all terrestrial ecosystems by providing ecosystem services through photosynthesis- derived compounds. Throughout the millennia, plant metabolism has diversified in the form of all plant secondary metabolites, ranging from metabolite groups such as terpenes to alkaloids to flavonoids. Many of these secondary metabolites are economically valued for their chemical, pharmaceutical and physical properties. The flavonoids are one of the largest groups and are known to provide competitional advantages and increase of survival of many plant species in extreme environments. One of the critical enzymes in the whole biosynthesis pathway of flavonoids is the dihydroflavonol 4- reductase (DFR). DFR regulates the formation of leucoanthocyanidins, predecessors of colourful anthocyanins. Anthocyanins are an economically significant group of molecules, especially for horticulturists and plant breeders, but also for nutritional and health scientists due to their potential health benefits. Dihydroflavonol 4-reductase is a much-studied enzyme due to its significant role in flavonoid biosynthesis and the economic interests of plant breeders and alike. Previous studies have expanded the knowledge of flavonoid biosynthesis and have identified several amino acid residues in the DFR structure affecting the substrate specificity of the enzyme and, consequently, the flower colours. However, only a single crystal structure model of the dihydroflavonol 4-reductase has been solved so far, originating from the grapevine Vitis vinifera. Although a single crystal structure can facilitate further structure-to-function studies associated with dihydroflavonol 4-reductase, further studies need to be carried out to shine a light on the functional basis of the enzyme. Therefore, this study aims to resolve petunia and gerbera dihydroflavonol 4-reductase crystal structures, expanding the knowledge of structural variations within the uncharted families of angiosperms, Solanaceae and Asteraceae. Several recombinant protein expression systems were utilised in my attempts to solve the crystal structure of the DFRs. These systems ranged from the bacterium Escherichia coli to yeast species such as Saccharomyces cerevisiae and Pichia pastoris, as well as the tobacco plant Nicotiana benthamiana. The genes encoding for Petunia wildtype DFRA, three mutants, and three Gerbera DFR variants were cloned to several expression vectors. Their presence and expression were identified using various genetic methodologies and enzymological assays. The expression of DFRs using an E. coli-based expression system was verified. However, the trials with E. coli were deemed unsuccessful due to the majority of the protein ending in inclusion bodies with no detectable activity. An alternative system using agroinfiltration of N. benthamiana was later utilised, as significant amounts were detected in the plant tissue extracts following the agrobacterial infiltration. Although the proteins were expressed in high quantities, no purification procedures have been established to provide plant tissue-extracted protein in crystallography-grade purity. With the protein supplied by a plant-based system and several small- scale purification steps, purified DFR enzymes could be utilised in crystallisation studies. Due to significant contamination by RuBisCO in the protein samples, alternative systems based on S. cerevisiae and Pichia pastoris were investigated, and a successful Pichia-based expression was established. Several sets of plasmids with variable expression systems were constructed in this study, facilitating future experiments into the dynamics and structure of dihydroflavonol 4-reductases. Ground-breaking techniques based on computational modelling were utilised to hypothesise the role of prior determined amino acid residues in enzyme catalysis and substrate recognition. Possible crystallisation-related issues originating from protein structure were approached using the same techniques, opening new windows and possibilities into determining the structure of Petunia hybrida and Gerbera hybrida dihydroflavonol 4-reductase structures using tools of protein engineering