Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation.
Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3-hydroxypropionate cycle for carbon dioxide assimilation. In this cycle the ATP-dependent carboxylations of acetyl-CoA and propionyl-CoA to malonyl-CoA and methylmalonyl-CoA, respectively, represent the key CO2 fixation reactions. These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and propionyl-CoA. The carboxylase was purified and characterized and the genes were cloned and sequenced. In contrast to the carboxylase of most other organisms, acetyl-CoA/propionyl-CoA carboxylase from M. sedula is active at 75 degrees C and is isolated as a stabile functional protein complex of 560 +/- 50 kDa. The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta). These subunits probably form an (alpha beta gamma)4 holoenzyme. It has a catalytic number of 28 s-1 at 65 degrees C and at the optimal pH of 7.5. The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. Acetyl-CoA/propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.
Hügler
Michael
M
Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, Germany.
Krieger
Robert S
RS
Jahn
Martina
M
Fuchs
Georg
G
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D015139:Blotting, Southern; D002245:Carbon Dioxide / Q000737:chemistry; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D044943:Fatty Acid Desaturases / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D008316:Malonyl Coenzyme A / Q000378:metabolism; D043586:Methylmalonyl-CoA Decarboxylase; D010088:Oxidoreductases; D010957:Plasmids; D021122:Protein Subunits; D016943:Sulfolobaceae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*
D000214:Acyl Coenzyme A; D021122:Protein Subunits; C015357:methylmalonyl-coenzyme A; D002245:Carbon Dioxide; D008316:Malonyl Coenzyme A; D001710:Biotin; D000105:Acetyl Coenzyme A; D010088:Oxidoreductases; C454539:malonyl-Coa reductase; D044943:Fatty Acid Desaturases; D002262:Carboxy-Lyases; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase; D000103:Acetyl-CoA Carboxylase; D043586:Methylmalonyl-CoA Decarboxylase