Characterization of a novel enzyme system for activation of acetone in sulfate-reducing bacteria

• Applicant: Professor Dr. Bernhard Schink
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Microbial Ecology and Applied Microbiology
• Term: from 2017 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 391398354

Whereas acetone-degrading aerobic, phototrophic, and nitrate-reducing bacteria activate their substrate by carboxylation to acetoacetate sulfate-reducing bacteria do not use acetone carboxylase, and acetoacetate is not a free intermediate in the pathway of acetone degradation. Studies with Desulfococcus biacutus in our lab showed that CO is a better co-substrate for acetone degradation than CO2, and that the activation reaction requires coenzyme A, ATP, and thiamine diphosphate as cofactors. According to our present working hypothesis, formyl-CoA is added to the carbonyl carbon of acetone in a thiamine diphosphate-dependent reaction to form 2-hydroxyisobutyryl-CoA which is subsequently isomerized to 3-hydroxybutyryl-CoA and oxidized to acetoacetyl-CoA which, after thiolytic cleavage, is further oxidized through the reversed Wood-Ljungdahl pathway. The planned project will concentrate on purification and characterization of the thiamine diphosphate-dependent 2-hydroxyisobutyryl-CoA synthase and on the formation of the putative co-substrate formyl-CoA. The study can be based on a completely sequenced and annotated genome of D. biacutus, proteomic identification of the enzymes involved in acetone metabolism, broad experience with heterologous expression of enzymes of this bacterium, and excellent chemical and analytical support (HPLC-MS etc.) by partner scientists at Konstanz university.

• DFG Programme: Research Grants
• Co-Investigator: Professor David Schleheck, Ph.D.