Chalcone 3-hydroxylase

The proposal Chalcone 3-hydroxylase studies a key enzyme in the biosynthesis of anthochlor pigments (chalcones and aurones), which provides yellow flower colour in a number of ornamental plants, form UV-honey guides in certain Asteraceae species and also show health-beneficial effects of chalcones in humans. Furthermore, chalcones are the immediate precursors for the formation of flavonoids, which are important compounds in plant-derived human diets and of isoflavonoid-based phytoalexins. Chalcone 3-hydroxylase (CH3H) belongs to the important class of cytochrome P450 dependent monooxygenases and catalyzes the introduction of a hydroxyl group in position 3 of chalcones. The reaction shows high similarity to the hydroxylation of flavonoids in position 3 but cannot be catalyzed by the prominent flavonoid 3- hydroxylase (F3H) despite its broad substrate specificity. The previous project P24331-B16 investigated differences in the structure of CH3H and F3H that could be relevant for their differences in functionality. Several regions determining functionality were identified but the effects could not be completely explained due to the lack of the crystal structure. The new project aims at elucidating the three-dimensional structure of CH3H to fully understand the structural differences determining the divergent functionality of CH3H and F3H. First, a strategy for producing large amounts of purified CH3H in a soluble form will be established. Then, crystallization experiments will be carried out to obtain the three-dimensional structure of the protein. Amino acids determining functionality will be identified and confirmed by site- directed mutagenesis. The project will provide the first crystal structure of a cytochrome P450 dependent enzyme of flavonoid pathway. This will enable a better understanding of the structure-function relationship of CH3H and further related enzymes of this important secondary metabolite pathway. In future, this will feed into the breeding of plants showing increased disease tolerance and/or improved attractiveness of plants and enhanced health beneficial effects to consumers.

The proposal 'Chalcone 3-hydroxylase' studied a key enzyme in the biosynthesis of anthochlor pigments (chalcones and aurones), which provides yellow flower colour in a number of ornamental plants, forms UV-honey guides in some Asteraceae species and also shows health-beneficial effects of chalcones in humans. Furthermore, chalcones are the immediate precursors for the formation of flavonoids, which are important compounds in plant-derived human diets and of isoflavonoid-based phytoalexins. Chalcone 3-hydroxylase (CH3H) belongs to the important class of cytochrome P450 dependent monooxygenases and catalyzes the introduction of a hydroxyl group in position 3 of chalcones. The reaction shows high similarity to the hydroxylation of flavonoids in position 3', but cannot be catalyzed by the prominent flavonoid 3'-hydroxylase (F3'H) despite its broad substrate specificity. The previous project P24331-B16 investigated differences in the structure of CH3H and F3'H that could be relevant for their functional differences. Several regions determining functionality were identified but the effects could not be completely explained due to the lack of the crystal structure. The new project therefore aimed at elucidating the three-dimensional structure of CH3H to fully understand the structural differences determining the divergent functionality of CH3H and F3'H. A process for the large-scale production in the bacterium Escherichia coli and multi-step purification of an engineered, soluble CH3H variant with full heme occupancy was successfully established, while a promising strategy for the large-scale production of the original membrane-bound CH3H in the yeast Pichia pastoris with subsequent purification and screening for suitable crystallization conditions was completed. A number of obstacles were successfully overcome (e.g. insufficient heme occupancy, formation of oligomers and inhomogeneous protein) requiring more time than originally assumed. In addition, we made significant progress in the identification of amino acids relevant for CH3H, F3'5'H and F3'H activity. The elucidation of the crystal structure will be performed in the frame of a follow-up project, which has already started. With the knowledge obtained in the project, more resistant, attractive and health-promoting plants can be grown in the future. The project enabled the performance of two doctoral theses, two master theses and three bachelor theses.