B-ring hydroxylation in the flavonoid pathway

The proposal B-Ring hydroxylation in the flavonoid pathway studies key enzymes in the biosynthesis of flavonoids and anthochlor pigments (chalcones and aurones), which provide yellow, red and blue flower colour in a number of ornamental plants, form UV-honey guides in certain Asteraceae species and also show health-beneficial effects 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 plant defence compounds. This is a follow up proposal to the expiring P29552-B29 project, that started the establishment of the first crystal structure of a cytochrome P450 dependent enzyme of the flavonoid pathway using chalcone 3-hydroxylase (CH3H) as model. Funding for this follow up project will be crucial to allowing the team to capitalize on the foundation work already performed during the previous project. CH3H 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. F3`5`H, in contrast, catalyze the introduction of two hydroxyl groups in positions 3` and 5`, thereby shifting the colour of resulting pigments from red to blue and violet. Previously, we successfully established methods for the large-scale production and purification of sufficient amounts of CH3H for crystallization experiments. The elucidated three-dimensional structure of CH3H is essential to fully understand the structural differences determining the divergent functionality of CH3H and F3`H and of the reaction mechanism of F3`5`H. 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 project 'B-Ring hydroxylation in the flavonoid pathway' was designed as a follow up proposal to P29552-B29, which started the establishment of the first crystal structure of a cytochrome P450 dependent monooxygenase (CYP) of the flavonoid pathway using the specialized CYP75B member chalcone 3-hydroxylase (CH3H) as a model. CH3H is closely related to the prominent flavonoid 3'-hydroxylase (F3'H) and a key enzyme in the biosynthesis of anthochlor pigments, which provide 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. The project aimed at enabling an understanding of the structure-function relationship of the CYPs determining the B-ring hydroxylation pattern of flavonoid structures (CH3H, F3'H, F3'5'H). The identification of the amino acids involved in substrate binding and their mode of action is of key interest as this allows targeted application in biotransformation, and breeding of plants with increased disease tolerance and/or improved aesthetic qualities. Procedures were established for the recombinant production of a soluble variant of CH3H and three cytochrome P450 reductases from different ornamental species with high yields, which allowed characterization of the purified enzyme and provided new insights into CH3H substrate specificity. Limited long-time stability of CH3H, however, so far impeded crystallization of the enzyme. Studies of F3'H from Malus sp. concentrated on the potential contribution to dihydrochalcone hydroxylation and its impact for watercore, an internal physiological disorder of apple. The results also contributed to the creation of the first genome edited poinsettias towards orange flower colour. For F3'5'H, amino acids essential for enzyme activity were identified for the first time. The project involved Heidi Halbwirth (expertise in flavonoid biosynthesis and hydroxylating enzymes), Oliver Spadiut (expertise in protein production and purification) and Christian Molitor (expertise in crystallization and modelling). This project led to seven publications in peer-reviewed journals.