Phd on topic: Oxidative C-H activation for C-C bond formation using the flavin dependent enzymes EasC and EasE


Research interest and scientific background: We have a long standing interest in flavoenzymes that oxidatively form C-C bonds such as the berberine bridge enzyme (1). The fungus Aspergillus japonicus produces the alkaloid chanoclavine through the oxidative cyclisation catalyzed by EasE, which is a homolog of the berberine bridge enzyme (2).

Affiliation: The student will work at the Institute of Biochemistry in enzymology and protein chemistry group at the Graz University of Technology under the supervision of Peter Macheroux. Cooperation partners in this project are Anton Glieder (biotechnology), Karl Gruber (structural biology) and Wolfgang Kroutil (biocatalysis). This project will be directly funded by CATALOX.

Hypothesis and objective: The project aims to investigate the mechanism of action of the enzymes, in particular of the flavoenzyme EasE, involved in the generation of chanoclavine. Thus, the primary goal will be the production of the active enzymes by recombinant biotechnology. Furthermore, the obtained proteins will be characterized with regard to their kinetic parameters and their substrate scope. Eventually, the enzymes will be employed to explore their utility in producing derivatives of the fungal alkaloid chanoclavine.

Experimental approaches: We will employ modern gene expression in selected host organisms, state-of-the-art protein purification methods as well as rapid reaction techniques to characterize our enzymes. In collaboration with Prof. Gruber, we will also attempt to solve the crystal structure of the proteins.

The student will strongly interact with other research groups of the doc.funds programs CATALOX and Molecular Metabolism (MOBILES), the Austrian Centre of Industrial Biotechnology (acib) and the research and training network NAWI Graz and BioTechMed Graz.

References:

  1. Winkler, A., Lyskowski, A., Riedl, S., Puhl, M., Kutchan, T., Macheroux, P., Gruber, K.: A concerted mechanism for berberine bridge enzyme, Nat. Chem. Biol., 2008, 4:739-741.
  2. Nielsen, C. A. F., Folly, C., Hatsch, A., Molt, A., Schröder, H., O’Connor, S. E., Naesby, M.: The important ergot alkaloid intermediate chanoclavine-I produced in the yeast Saccharomyces cerevisiae by the combined action of EasC and EasE from Aspergillus japonicus, Microb. Cell Fact., 2014, 13:95-105.