Understanding and engineering biomolecular recognition in Rieske oxygenases

Project description

We are interested in deciphering the molecular basis for the high specificity of Rieske oxygenases and their electron carrier proteins, so-called ferredoxins.

Biotechnological processes offer clean and sustainable conditions for challenging chemical reactions. Biocatalysts used in Biotechnology are referred to as enzymes. A special class of such enzymes include oxidoreductases which catalyze selective redox reactions. Redox biocatalysis is already a very successful, environmentally friendly technology for the synthesis of high-value fine chemicals and pharmaceuticals. Nevertheless, the synthetic potential of many enzymes has not been fully exploited yet, as their structure and thus their functionality and production is very complex. Furthermore, many oxidoreductases depend on a sophisticated electron transport chain that includes the fine-tuned interplay of multiple proteins. Thus, the proposed project investigates how this electron transport chain can be better understood and, on the long-term, simplified to broaden the applicability of multi-component oxidoreductases for the production of various pharmaceuticals, such as the indinavir (CRIXIVAN) precursor indandiol. In particular, ferredoxins will be investigated, which occur as universal electron transfer proteins in the metabolism of numerous organisms and display the same function within those. Although ferredoxins from different organisms have the same function, they differ considerably in their structure. As a result, only ferredoxins with the same or very similar structure can interact with their oxidoreductase and transfer electrons to it. Only if this interaction and thus the electron transport is successful, the oxidoreductase catalyzes the desired chemical reaction.

The main goal of this project is to increase the understanding of the essential aspects determining an efficient electron transfer based on specific protein-protein interactions between the respective partners. These key factors may be the efficiency of the electron transfer and consumption by the enzyme-catalyzed conversion on the one hand, but also the identification of key limiting factors in terms of low expression yields and instability on the other hand. On the basis of a structural analyses and molecular dynamics simulations, we will identify key residues determining the protein-protein interactions between the electron donor and acceptor proteins. The student will thus start with a structural analysis of the model proteins and will then construct a library of oxygenase and ferredoxin variants, using saturation mutagenesis at several combined positions on the surface of the proteins. Screening or selection will be used to identify improved variants, of which the changes on the amino acid level will be deeper investigated by several methods (MST, CV, ITC). Finally, the biocatalytic properties will be studied and the improved variants will be applied for the synthesis of model products.

Organization

The master research project will be conducted in the Department of Chemical and Pharmaceutical Biotechnology, in the research group of Dr. Sandy Schmidt. Our laboratory is well equipped for molecular biology, enzymology, analytics, protein engineering and biocatalysis work. We offer a stimulating research environment for a student interested in these topics.

Contact

For more information, please contact Dr. Sandy Schmidt.
Chemical and Pharmaceutical Biology, GRIP
Building 3215, room 9.29
Antonius Deusinglaan 1
9713 AV Groningen