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

  • Rauch-Diamantis, Gernot, (Co-Investigator (CoI))
  • Lehsl, Annemarie, (Co-Investigator (CoI))
  • Kayer, Heidemarie, (Co-Investigator (CoI))
  • Macheroux, Peter (Principal Investigator (PI))

Project: Research project

Description

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.
StatusFinished
Effective start/end date1/09/0431/08/07