The sodium ion translocating glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans : cloning and function of the genes forming a second operon[Note 4. Enzymes: Acetyl‐CoA carboxylase (EC 6.4.1.2); glutaconyl‐CoA decarboxylase (EC 4.1.1.70); ...]

Glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans (clostridal cluster IX), a strict anaerobic inhabitant of animal intestines, uses the free energy of decarboxylation (ΔG°′ ≈ −30 kJ mol −1 ) in order to translocate Na + from the inside through the cytoplasmic membrane. The proton, which is required for decarboxylation, most probably comes from the outside. The enzyme consists of four different subunits. The largest subunit, α or GcdA (65 kDa), catalyses the transfer of CO 2 from glutaconyl‐CoA to biotin covalently attached to the γ‐subunit, GcdC. The β‐subunit, GcdB, is responsible for the decarboxylation of carboxybiotin, which drives the Na + translocation (approximate K m for Na + 1 mM), whereas the function of the smallest subunit, δ or GcdD, is unclear. The gene gcdA is part of the ‘hydroxyglutarate operon’, which does not contain genes coding for the other three subunits. This paper describes that the genes, gcdDCB , are transcribed in this order from a distinct operon. The δ‐subunit (GcdD, 12 kDa), with one potential transmembrane helix, probably serves as an anchor for GcdA. The biotin carrier (GcdC, 14 kDa) contains a flexible stretch of 50 amino acid residues (A26–A75), which consists of 34 alanines, 14 prolines, one valine and one lysine. The β‐subunit (GcdB, 39 kDa) comprising 11 putative transmembrane helices shares high amino acid sequence identities with corresponding deduced gene products from Veillonella parvula (80%, clostridial cluster IX), Archaeoglobus fulgidus (61%, Euryarchaeota), Propionigenium modestum (60%, clostridial cluster XIX), Salmonella typhimurium (51%, enterobacteria) and Klebsiella pneumoniae (50%, enterobacteria). Directly upstream of the promoter region of the gcdDCB operon, the 3′ end of gctM was detected. It encodes a protein fragment with 73% sequence identity to the C‐terminus of the α‐subunit of methylmalonyl‐CoA decarboxylase from V. parvula (MmdA). Hence, it appears that A. fermentans should be able to synthesize this enzyme by expression of gctM together with gdcDCB, but methylmalonyl‐CoA decarboxylase activity could not be detected in cell‐free extracts. Earlier observations of a second, lower affinity binding site for Na + of glutaconyl‐CoA decarboxylase (apparent K m 30 mM) were confirmed by identification of the cysteine residue 243 of GcdB between the putative helices VII and VIII, which could be specifically protected from alkylation by Na + . The α‐subunit was purified from an overproducing Escherichia coli strain and was characterized as a putative homotrimer able to catalyse the carboxylation of free biotin.