Fermentation of 4-aminobutyrate by Clostridium aminobutyricum: cloning of two genes involved in the formation and dehydration of 4-hydroxybutyryl-CoA

Clostridium aminobutyricum ferments 4-aminobutyrate via succinic semialdehy de, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA and crotonyl-CoA to acetate and butyrate. The genes coding for the enzymes that catalyse the interconversi on of these intermediates are arranged in the order abfD (4-hydroxybutyryl- CoA dehydratase), abfT (4-hydroxybutyrate CoA-transferase), and abfH (NAD-d ependent 4-hydroxybutyrate dehydrogenase). The genes abfD and abfT were clo ned, sequenced and expressed as active enzymes in Escherichia coli. Hence t he insertion of the [4Fe-4S]clusters and FAD into the dehydratase required no additional specific protein from C. aminobutyricum. The amino acid seque nces of the dehydratase and the CoA-transferase revealed close relationship s to proteins deduced from the genomes of Clostridium difficile, Porphyromo nas gingivalis and Archaeoglobus fulgidus. In addition the N-terminal part of the dehydratase is related to these of a family of FAD-containing mono-o xygenases from bacteria. The putative assignment in the databank of Ca2 (Or fZ) from Clostridium kluyveri as 4-hydroxybutyrate CoA-transferase, which i s thought to be involved in the reductive pathway from succinate to butyrat e, was confirmed by sequence comparison with AbfT (57% identity). Furthermo re, an acetyl CoA:4-hydroxybutyrate CoA-transferase activity could be detec ted in cell-free extracts of C, kluyveri. In contrast to glutaconate CoA-tr ansferase from Acidaminococcus fermentans, mutation studies suggested that the glutamate residue of the motive EXG, which is conserved in many homolog ues of AbfT: does not form a CoA-ester during catalysis.