Recombinant core proteins of aurone synthases

Aurones are yellow plant pigments which are biochemically related to flavonoids. Apart from their physiological function as attractants of pollinators in flowers, aurones are of great pharmacological and biotechnological interest. Two types of aurones, 4-hydroxyaurones and 4-deoxyaurones, exist differing by the presence or absence of a hydroxyl group in position 4. Formation of 4-hydroxyaurones has been studied in detail in the snapdragon (Antirrhinum majus). Aureusidine synthase (AmAS1), a polyphenol oxidase (PPO) homologue exhibiting monophenolase activity, was shown to be responsible. This introduced a novel enzyme type in the flavonoid pathway and provided a popular example for the involvement of plant PPOs in anabolic reactions. 4-Deoxyaurone was assumed to occur in a similar way and has therefore been neglected for a long time. We have recently demonstrated that the biosynthesis of 4-deoxyaurones differs from that of 4-hydroxyaurones in various aspects including the enzyme type, the substrate specificity and subcellular location. This project aims at studying open questions regarding the enzymes involved in 4-deoxyaurone biosynthesis. With the exception of CgAUS1, the native corresponding enzymes are not easily accessible in amount and qualities sufficient to allow comparative studies of substrate specificities and enzyme structures. We therefore aim to use recombinant enzymes. Because aurone synthases like PPOs are produced as inactive proenzymes (synonym latent enzymes) and activated by proteolytic cleavage releasing an active core enzyme, we aim at engineering aurone synthase cDNA clones which allow to mimic proteolytic cleavage in planta by artificially introduced PreScission protease cleavage sites. Thus, this project lays the foundation for facilitated production of recombinant active core enzymes of PPOs and aurone synthases and will enable future studies on the structure and function of this enzyme class. Recombinant active core proteins will be used for a detailed characterization of CgAUS2 in comparison to CgAUS1 and AmAS1 and unspecific PPOs. Recombinant and latent CgAUS2 will be purified in large scale and crystallization attempts will be started to compare the structures of CgAUS2, which we assume to exhibit broad substrate specificity, with the chalcone selective CgAUS1, for which we obtained already crystal structures. Finalization of this project part will depend on the transferability of the optimized crystallization conditions from CgAUS1 to CgAUS2. To date, only two cDNA clones encoding aurone synthases (AUS) from Coreopsis grandiflora, CgAUS1 and CgAUS2, and the AmAS1 from A. majus are available for studying differences in the aurone formation. The project will therefore isolate further cDNA clones from aurone forming species to analyze the distribution of the two ways of aurone formation, phylogenetic relationships and sequence similarities.

Aurones are yellow flower pigments and biochemically related to the flavonoids. They serve to attract pollinators, but are also of pharmacological and biotechnological interest. There are two basic types, 4-hydroxyaurones and 4-deoxyaurones, which differ in the hydroxylation pattern at the position 4 in the A-ring. The biosynthesis of the 4-hydroxyaurones in snapdragon (Antirrhinum majus) has been thoroughly investigated. The 4-deoxyaurones, on the other hand, has received less attention for a very long time, since their biosynthesis was assumed to take place analogously. However, their formation differs in essential points from that of 4-hydroxyaurones. The so-called aurone synthases are the key enzyme in the biosynthesis of aurones. They belong to the important class of polyphenol oxidases. This project studied different aspects of 4-deoxyaurone biosynthesis. Since at the beginning of the project only two cDNA clones from Coreopsis grandiflora were known, in addition to the enzyme from the snapdragon, an important focus of the work was the identification and characterization of previously unknown aurone synthases (AUS) from other plants. As part of this project, further cDNA clones were identified and isolated from the yellow flowering bedding plants Cosmos, Coreopsis, Dahlia and Bidens. In addition, sequences of two polyphenol oxidases from the banana were investigated. Another focus was the production of recombinant polyphenol oxidases in latent and active forms. It could be shown that the heterologous expression of a partial structure is sufficient for the production of an active protein. Subsequently, the importance of polyphenol oxidases for biosynthetic pathways in various plants was investigated. For this purpose, combined biochemical, molecular biological and analytical investigations were carried out on the one hand, and bioinformatic methods and transcriptome analyses of various yellow-flowered ornamental plants, as well as of apples and bananas, on the other. The biosynthesis of aurones or of anthochlor pigments in general was investigated in various ornamental plants and expanded to include polyphenol oxidases in defense reactions in apples and bananas. In cooperation with the University of Ljubljana, the involvement of polyphenol oxidases in watercore affecte apples was investigated. The project has layed the foundation for future investigations of the functions and structures of the important enzyme class, the polyphenols, using recombinant proteins.