Functional characterization of cytochrome P450 dependent monooxygenases from euphorbia pulcherrima

Abstract

Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3', 5'-hydroxylase (F3'5'H) are cytochrome P450 dependent monooxygenases and part of the flavonoid pathway. Flavonoids are a class of secondary metabolites in plants including many flower pigments. One class of flavonoids are anthocyanins which are red, blue and violet plant pigments. F3'H and F3'5'H determine the hydroxylation pattern on the B-ring of anthocyanins and flavonoids in general. F3'H catalyzes hydroxylation in position 3' and F3'5'H catalyzes hydroxylation in positions 3' and 5'. These hydroxylation patterns lead to different types of anthocyanins and therefore to different coloring of the plant. Hydroxylation in position 3' catalyzed by F3'H leads to cyanidin-based anthocyanins and dark red color. Hydroxylation in positions 3' and 5' catalyzed by F3'5'H leads to delphinidin-based anthocyanins and blue coloring. This thesis focuses on the F3'H and F3'5'H of Euphorbia pulcherrima cultivar 'Christmas Feelings'. Euphorbia pulcherrima is commonly known as poinsettia or Christmas Flower. The cultivar was developed by Selecta Klemm GmbH & Co. KG. Characteristic features of Euphorbia pulcherrima are the big red bracts. The red coloration of the bracts is due to the high amounts of cyanidin-based anthocyanins. This indicates the presence of an active F3'H catalyzing hydroxylation in position 3' in the flavonoid pathway. Euphorbia pulcherrima in general does not show blue bract coloration. However, transcription studies have shown the presence of a putative gene of F3'5'H. But the corresponding recombinant protein F3'5'H turned out to be inactive. The aim of this master thesis was to characterize the active F3'H and the inactive F3'5'H of Euphorbia pulcherrima with a focus on identifying the differences between F3'H and F3'5'H in general. In order to see which amino acids are important for functional activity of the enzyme, different active F3'5'Hs were aligned with the one of Euphoria pulcherrima. Additionally, homology models were made to see which amino acids are involved in substrate binding. Three amino acid residues were chosen for exchange and site directed mutagenesis was performed to see the impact of these amino acids on substrate binding. For F3'H, the aim was the recombinant expression of the enzyme in E. coli strains. Furthermore, a method of purification for this enzyme was developed with the prospect of crystallization and determination of the structure of the protein.