3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation.
A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO2 fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 micromol min(-1) mg protein(-1). The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA --> 3-hydroxypropionyl-CoA + AMP + PP(i). The enzyme was purified 95-fold to a specific activity of 18 micromol min(-1) mg protein(-1) from autotrophically grown M. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli, and the His-tagged protein was purified. Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula, supporting its proposed role during CO2 fixation in this archaeon and possibly other members of the Sulfolobaceae family.
Alber
Birgit E
BE
Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, Germany. alber.8@osu.edu
Kung
Johannes W
JW
Fuchs
Georg
G
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism*; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019344:Lactic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D008954:Models, Biological; D015394:Molecular Structure; D013379:Substrate Specificity; D016943:Sulfolobaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D000214:Acyl Coenzyme A; D019843:Archaeal Proteins; D002245:Carbon Dioxide; C009061:propionyl-coenzyme A; D019344:Lactic Acid; C031601:hydracrylic acid; D008291:Malate Dehydrogenase; D003066:Coenzyme A Ligases; D003065:Coenzyme A