Radical reactions in fermentation pathways of nitrogenous compounds

• Applicant: Professor Dr. Wolfgang Buckel
• Co-Applicant: Professor Dr. Bernhard Golding
• Subject Area: Biochemistry
• Term: from 1999 to 2007
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5178032

The mechanisms of the coenzyme B12-dependent glutamate and 2-methyleneglutarate mutase, as well as the [4Fe-4S]cluster- and FAD-dependent 4-hydroxybutyryl-CoA dehydratase, will be investigated. It is proposed that catalysis by all three clostridial enzymes is a consequence if hydrogen abstraction. Kinetic isotope effects and newly designed inhibitors will be used to determine the rate limiting steps. The use of such inhibitors may cause the radical intermediates to persist in sufficient concentrations and enable their analysis by EPR and ENDOR spectrocsopy. Therefore, a series of substrates, have to be synthesised. The formation of a methyl group by each enzyme will analysed by the chiral acetate methodology. Determination of the steric course of the b,g-elimination of water from 4-hydroxybutyryl-CoA to crotonyl-CoA may tell whether the reaction occurs in a stepwise or concerted manner. Mutagenesis of essential amino acid residues, identified by the emerging three-dimenional structures, should help to define the role of the proteins in these catalyses.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1071: Radikale in der enzymatischen Katalyse
• International Connection: United Kingdom