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 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.
Gerhardt
A
A
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Germany.
Cinkaya
I
I
Linder
D
D
Huisman
G
G
Buckel
W
W
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D015256:Coenzyme A-Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
C101347:4-hydroxybutyryl-coenzyme A; D000214:Acyl Coenzyme A; D005680:gamma-Aminobutyric Acid; C072231:4-hydroxybutyrate CoA-transferase; D015256:Coenzyme A-Transferases; C066370:4-hydroxybutyryl-CoA dehydratase; D006836:Hydro-Lyases