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

• Published in: Archives of microbiology 2000-09, Vol.174 (3), p.189-199
• Main Authors: GERHARDT, A, CINKAYA, I, LINDER, D, HUISMAN, G, BUCKEL, W
• Format: Article
• Language: English
• Subjects: 4-aminobutyric acid; 4-hydroxybutyryl-CoA; abf gene; Acyl Coenzyme A - metabolism; Amino Acid Sequence; Bacteriology; Biological and medical sciences; Cloning, Molecular; Clostridium - enzymology; Clostridium - genetics; Clostridium aminobutyricum; Coenzyme A-Transferases - chemistry; Coenzyme A-Transferases - genetics; Coenzyme A-Transferases - metabolism; Fermentation; Fundamental and applied biological sciences. Psychology; gamma-Aminobutyric Acid - metabolism; Genes, Bacterial; Hydro-Lyases - chemistry; Hydro-Lyases - genetics; Hydro-Lyases - metabolism; Metabolism. Enzymes; Microbiology; Molecular Sequence Data; Phylogeny; Sequence Alignment
• ISSN: 0302-8933; 1432-072X
• DOI: 10.1007/s002030000195

Clostridium aminobutyricum ferments 4-aminobutyrate via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA and crotonyl-CoA to acetate and butyrate. The genes coding for the enzymes that catalyse the interconversion of these intermediates are arranged in the order abfD (4-hydroxybutyryl-CoA dehydratase), abfT (4-hydroxybutyrate CoA-transferase), and abfH (NAD-dependent 4-hydroxybutyrate dehydrogenase). The genes abfD and abfT were cloned, sequenced and expressed as active enzymes in Escherichia coli. Hence the insertion of the [4Fe-4S]clusters and FAD into the dehydratase required no additional specific protein from C. aminobutyricum. The amino acid sequences of the dehydratase and the CoA-transferase revealed close relationships to proteins deduced from the genomes of Clostridium difficile, Porphyromonas gingivalis and Archaeoglobus fulgidus. In addition the N-terminal part of the dehydratase is related to those of a family of FAD-containing mono-oxygenases from bacteria. The putative assignment in the databank of Cat2 (OrfZ) from Clostridium kluyveri as 4-hydroxybutyrate CoA-transferase, which is thought to be involved in the reductive pathway from succinate to butyrate, was confirmed by sequence comparison with AbfT (57% identity). Furthermore, an acetyl-CoA:4-hydroxybutyrate CoA-transferase activity could be detected in cell-free extracts of C. kluyveri. In contrast to glutaconate CoA-transferase from Acidaminococcus fermentans, mutation studies suggested that the glutamate residue of the motive EXG, which is conserved in many homologues of AbfT, does not form a CoA-ester during catalysis.