Crystallization of type III polyketide synthases from Hypericaceae and Gentianaceae, immunodetection of CYP736A163 involved in aucuparin biosynthesis in Rosaceae, and functional characterisation of prenyltransferases involved in hyperforin biosynthes

Plants produce a variety of complex chemical molecules that originate from secondary metabolism, helping them adapt to their environment and ensuring their survival, though the precise mechanism of action of individual components is often unknown. Due to their taste and nutritional value and their biological activity, plants or plant preparations are used by humans either as food, as is the case with members of the Rosaceae family (apples, pears, stone fruits, etc.), or as medicine, such as preparations from Hypericum perforatum. Enzymes are crucial for the biosynthesis of these secondary metabolites. Therefore, this work investigates various enzymes involved in the formation of secondary compounds in plants. One objective is the immunolocalization of CYP736A, an ER- anchored membrane protein involved in phytoalexin biosynthesis in Rosaceae species, such as Sorbus aucuparia cell suspension cultures. Phytoalexins are chemical compounds whose synthesis is prompted by an external stimulus, such as contact with a pathogen. This investigation aims to explore the possible relocation of CYP736A after pathogenic attack and its potential involvement in a metabolon implicated in phytoalexin biosynthesis. A specially produced peptide antibody, targeting a defined, surface exposed region of the protein, was used for precise identification of CYP736A. It was able to specifically detect the recombinant protein but failed to detect the protein extracted from plant material in Western blots. Moreover, the cell suspension cultures have to be reconsidered for these experiments, prompting the discussion of alternative approaches. Additionally, three type III polyketide synthases were examined regarding their three-dimensional structure using X-ray crystallography. These enzymes play an important role in the biosynthesis of secondary metabolites, providing the backbone for further reactions to more complex molecules. The two type III PKS from Gentianaceae, CeBPS and ScBPS, can process large substrates, suggesting a novel catalytic mechanism. The type III PKS from Hypericum polyphyllum is a novel bifunctional PKS, whose structural elucidation should contribute to the understanding of the cyclization mechanisms. However, in this work, it was not possible to crystallize CeBPS and ScBPS. Furthermore, the crystals of HpBFPKS were unsuitable for elucidating the protein structure. Finally, during this work, a prenyltransferase, PT24 from Hypericum perforatum was characterized, and its kinetic parameters were determined. It was found that the protein, depending on the substrate, is involved in one of the initial steps of the biosynthesis of hyperforin and adhyperforin as well as secohyperforin and adsecohyperforin. The identification and characterization of this enzyme contribute to understanding the biosynthetic pathway of these PPAPs. This may enable the synthesis of analogues with fewer side effects, thereby enhancing therapeutic benefits