The sodium ion translocating glutaconyl-CoA decarboxylase from Acidaminococcus fermentans: cloning and function of the genes forming a second operon

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans (clostridal cl uster IX), a strict anaerobic inhabitant of animal intestines, uses the fre e energy of decarboxylation (Delta G degrees' approximate to -30 kJ mol(-1) ) in order to translocate Nat from the inside through the cytoplasmic membr ane. The proton, which is required for decarboxylation, most probably comes from the outside. The enzyme consists of four different subunits. The larg est subunit, alpha or GcdA (65 kDa), catalyses the transfer of CO2 from glu taconyl-CoA to biotin covalently attached to the gamma-subunit, GcdC. The b eta-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, delta 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 delta-sub unit (GcdD, 12 kDa), with one potential transmembrane helix, probably serve s as an anchor for GcdA. The biotin carrier (GcdC, 14 kDa) contains a flexi ble stretch of 50 amino acid residues (A26-A75), which consists of 34 alani nes, 14 prolines, one valine and one lysine. The beta-subunit (GcdB, 39 kDa ) comprising 11 putative transmembrane helices shares high amino acid seque nce identities with corresponding deduced gene products from Veillonella pa rvula (80%, clostridial cluster IX), Archaeoglobus fulgidus (61%, Euryarcha eota), Propionigenium modestum (60%, clostridial cluster XIX), Salmonella t yphimurium (51%, enterobacteria) and Klebsiella pneumoniae (50%, enterobact eria). 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 a-subunit of methylmalonyl-Coa decarboxy lase 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 si te for Na+ of glutaconyl-CoA decarboxylase (apparent K-m 30 mM) were confir med by identification of the cysteine residue 243 of GcdB between the putat ive helices VII and VIII, which could be specifically protected from alkyla tion by Nat. The cu-subunit was purified from an overproducing Escherichia coli strain and was characterized as a putative homotrimer able to catalyse the carboxylation of free biotin.