The biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase from Fusobacterium nucleatum (subsp. nucleatum). Comparison with the glutaconyl-CoA decarboxylases from gram-positive bacteria.
Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5 M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na+ (Km = 3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent Km for Na+ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, alpha (m = 65 kDa), beta (33 kDa), gamma (19 kDa), and delta (16 kDa) with the functions of a carboxy transferase (alpha), a carboxy lyase (beta and probably delta) and a biotin carrier (gamma). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the alpha- and the biotin containing subunits of the three decarboxylases and that from Peptostreptococcus asaccharolyticus could be detected on Western blots.
Beatrix
B
B
Laboratorium für Mikrobiologie, Philipps-Universität, Marburg, Federal Republic of Germany.
Bendrat
K
K
Rospert
S
S
Buckel
W
W
D001693:Biological Transport, Active; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005673:Fusobacterium / Q000201:enzymology*; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*
D010743:Phospholipids; D001710:Biotin; D012964:Sodium; D002262:Carboxy-Lyases; C023060:glutaconyl CoA decarboxylase