FWF - Mechanismus der Choris - Mechanistic and structural studies on chorismate synthase

The shikimate pathway is a major metabolic route for the biosynthesis of aromatic compounds in bacteria, fungi and plants. Owing to the lack of the shikimate pathway in animals, the enzymes of the pathway are interesting targets for the development of novel antibiotics, fungicides and herbicides as exemplified by the broad-spectrum herbicide glyphosate, which inhibits 5-enolpyruvylshikimate 3-phosphate synthase, the sixth enzyme of the pathway. The next enzyme, chorismate synthase catalyzes an anti-1.4-elimination reaction which also requires a reduced FMN cofactor. Recently, the three-dimensional structure of the protein was solved, demonstrating that the substrate and reduced FMN are in close proximity. The availability of a high-resolution structure provides the opportunity to investigate the mechanism of catalysis and the structure-function relationships in the active site. This investigation will encompass the generation of mutant proteins by site-directed mutagenesis and characterization of their catalytic properties employing kinetic and spectroscopic techniques. This approach will lead to a detailed understanding of the role of amino acid residues in the active site and will provide further insight into this unique catalytic reaction. A second focus of our research effort will address the unsolved question of how reduced FMN is delivered to chorismate synthase. In the case of the so-called monofunctional enzymes, it appears that a NADPH-dependent oxidoreductase generates reduced FMN, which is then passed on to the active site of chorismate synthase. This latter process is believed to occur in a protein-protein complex. We will attempt to isolate this complex from the model organism Bacillus subtilis and identify the hitherto unknown oxidoreductase. The identification of the interacting oxidoreductase will also enable us to search for homologs in other species and address the question of their phylogenetic relationship. On the other hand, bifunctional chorismate synthases from fungal species can utilize NADPH directly to reduce the FMN cofactor to the active redox state. Based on the three-dimensional structure of the enzyme we will determine the structural elements that bring about bifunctionality and confirm our working hypothesis by means of a set of mutagenesis experiments aiming to convert bifunctional chorismate synthase to a monofunctional enzyme and vice versa.