3-Hydroxypropionyl-Coenzyme A Synthetase from Metallosphaera sedula , an Enzyme Involved in Autotrophic CO 2 Fixation

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Abstract
A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO 2 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 μmol 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 μmol 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 CO 2 fixation in this archaeon and possibly other members of the Sulfolobaceae family.