Structure and function of fumarate-adding glycyl radical enzymes: biochemistry, modelling and application

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This proposal intends to investigate the biochemistry and reactions mechanisms of a class of novel enzymes which catalyse the radical-based addition of fumarate to a number of highly recalcitrant compounds, like hydrocarbons. These fumarate-adding enzymes (FAE) form a subclass of the glycyl radical enzymes which are highly oxygen-sensitive and need to be activated to the radical state by special activating enzymes. The proposed project is highly interactive between the German and Polish partners and will follow the following major aims: (A) mutagenesis studies to identify the functions of particular active site constituents and to extend the available product spectra, (B) characterization of other FAE after recombinant expression to investigate the respective accommodations to other substrates and including crystallization of various FAE to extend the structural knowledge basis on these enzymes, (C) studies on the activating enzyme to establish in-vitro activation, (D) multiscale high-level computational investigations of the FAE reaction mechanism, backed up with experimental input, and (E) developing methods to apply FAE and their mutant variants for biotechnological production of novel compounds. The project will employ state-of-the-art techniques from the field of molecular biology and enzymology combined with molecular modelling and engineering of biotechnological processes. The results will improve current understanding of bioremediation processes of environmental contaminations caused by fossil fuel and chemical industries and will deliver fundamental biochemical data which will improve the use of microorganisms in bioremediation of hydrocarbons. The elucidation of mechanism for the whole class of FAE will provide new insights into a novel chemical reaction principle exploited by these enzymes. Finally, studies of FAE will also provide new tools of biotechnological processes to synthesized fine chemicals and substrates for biodegradable polymers.