Studies on the reaction mechanism of corrinoid-reducing metallo-ATPases

• Applicant: Dr. Sandra Studenik
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 288495154

Protein bound corrinoid cofactors play an essential role as methyl group carriers in the C1-metabolism of anaerobes. To bind methyl groups, the corrinoid cofactor has to be in its super-reduced [CoI]-state, which is highly sensitive to autoxidation; as a consequence, the inactive [CoII]-state can be formed. Hence, a "re-activation" (i. e. reduction) is required, which is in general mediated by metallo-ATPases. This project will mainly focus on the ATP-dependent activation of protein-bound corrinoid cofactors of O-demethylase enzyme systems of Acetobacterium dehalogenans. In this microorganism, corrinoid reduction is mediated by the activating enzyme (AE) by increasing the midpoint potential of the [CoII]/[CoI]-couple from <-550 mV to about -320 mV in the presence of ATP and titanium(III) citrate as artificial electron donor. AE is a member of the COG3894 protein family, has a size of about 65 kDa and contains a [2Fe-2S]-cluster. The physiological electron donor for the corrinoid reduction as well as the enzymatic reaction, which leads to its reduction, will be identified. To gain more information on the reaction mechanism of the ATP-dependent corrinoid reduction, the structure of the protein complex consisting of AE and the corrinoid protein (CP) in the [CoII]-state, will be elucidated by protein crystallography. The redox potential of the corrinoid in CP will be studied in the presence and absence of AE or ATP by redox-titration coupled to UV/visible spectroscopy. Furthermore, the function of three other COG3894 proteins, which are encoded in the genome of A. dehalogenans and possibly act as corrinoid-reducing enzymes, will be elucidated. Therefore, the corresponding genes will be transformed in Escherichia coli and the gene products will be characterized with respect to their predicted function. The studies on the ATP-dependent corrinoid activation will be extended to Desulfitobacterium hafniense. Desulfitobacteria are non-acetogenic anaerobic microorganisms that are mainly described as reductively dehalogenating bacteria. They also harbor O-demethylase enzyme systems, which allow for the utilization of methylated substrates. The experiments will aim to contribute to the knowledge on the mechanism of thermodynamically unfavorable electron transfer reactions, which are driven by the hydrolysis of ATP.

• DFG Programme: Research Grants