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

A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO2 fixation pathway for the thermoacidophilic crenarchaeonMetallosphaera 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 + PPi . The enzyme was purified 95-fold to a specific activity of 18 μmol min−1 mg protein−1 from autotrophically grownM. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome ofSulfolobus tokodaii ; similar genes were found inS. solfataricus andS. acidocaldarius . The gene was heterologously expressed inEscherichia coli , and the His-tagged protein was purified. Both the native enzyme fromM. sedula and the recombinant enzyme fromS. 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 grownM. sedula , supporting its proposed role during CO2 fixation in this archaeon and possibly other members of theSulfolobaceae family.