Research projects

Research projects of current students

Fredrick E. Lyatuu
Nationality: Tanzanian
M.Sc. Thesis:
“Synthesis of Potential Nitro- and Fluoro-Inhibitors of Isocitrate Lyase (ICL) and 2-Methylcitrate Dehydratase”. (2004) Newcastle University, UK.
Research Group:
AG Buckel, Department of Microbiology, Philipps University and Max Planck Institute for Terrestrial Microbiology Marburg
Start of PhD Thesis: October, 2008
Research project:
Probing the Active Sites of Coenzyme B12 Dependent Glutamate Mutase and 2-Methyleneglutarate Mutase.
Coenzyme B12 (adenosylcobalamin) is the structurally most complex cofactor used by nature for enzymatic catalysis. The cobalt-carbon sigma-bond of the cofactor is integral to its mode of action.There are more than 10 known coenzyme B12 dependent biological reactions. During these reactions, the primary organic radical (5'-deoxyadenosyl) and the paramagnetic species; cob(II)alamin arise from homolysis of the Co-C bond when the coenzyme is bound to a partner enzyme in the presence of a substrate molecule. The former initiates a series of otherwise ‘impossible’ reactions, whereby protein-bound radicals are formed from a substrate molecule and enable 1,2-group migration leading to carbon skeleton rearrangements and transposition of amino groups in mutases and elimination of water or ammonia in eliminases. We are using probe molecules with wide range of spectroscopic techniques and X-ray crystallography to understand the mechanisms of coenzyme B12 dependent glutamate mutase and 2-methyleneglutarate mutase catalysed reactions. We have completed the synthesis of some inactivators which are stable analogues of radical intermediates in glutamate mutase fragmentation-recombination mechanism for use in our proposed mechanistic studies. In principle, these compounds could either competitively inactivate the enzyme or form adduct radicals with 5'-deoxyadenosyl radical and can be characterised in enzyme active site by UV/Visible and EPR spectroscopes. The cob(II)alamin participation in modulating intermediate radicals stabilities in mutases (conductor) as hypothesized by Buckel at. al. 2005, can also be demonstrated by EPR spectroscopy from these radical adducts.
2-Methyleneglutarate mutase protein is possibly too labile for crystallisation, because hitherto all attempts failed. Therefore, we wish to crystallise the subunit domains of the protein, which could be obtained by either protease digestion or by expression of mutated 2-methyleneglutarate mutase encoding genes. The structure of the whole enzyme will be obtained by electron density mapping