Mechanism of radical-mediated dehydration reactions in anaerobic bacteria

• Applicant: Professor Dr. Wolfgang Buckel
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2006 to 2009
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 28545261

Usual dehydratases catalyse the elimination of water from 3-hydroxyacyl-CoA. The electron-withdrawing thiol ester acidifies the ¿-hydrogen to pK <21 and enables its removal by basic amino acid side chains. Anaerobic bacteria, however, ferment amino acids via 2- or 4-hydroxyacyl-CoA, whose dehydrations involve the abstraction of a ß-hydrogen, which is ostensibly non-acidic (pK ca. 40). We have proposed mechanisms, in which one-electron transfer generates enoxy radicals with much lower pK-values and in which the elimination of the hydroxy groups involves ketyl radical anions. We want further elucidate these unprecedented mechanisms by site-directed mutagenesis of three participating enzymes: 2-Hydroxyisocaproyl-CoA dehydratase from Clostridium difficile, which is activated by one-electron reduction mediated by ATP, ferredoxin and an archerase or activator. The electron is further transferred to the substrate generating an enoxy radical. In contrast the action of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum requires transient one electron-oxidation of the substrate to an enoxy radical. In addition we plan to analyse the specificity and stereochemistry of the substrates of these unusual enzymes, which will substantiate conclusions drawn from X-ray crystallography.

• DFG Programme: Research Grants