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- Title
Discovery and characterization of a Coenzyme A disulfide reductase fromPyrococcus horikoshii.
- Authors
Harris, Dennis R.; Ward, Donald E.; Feasel, Jeremy M.; Lancaster, Kyle M.; Murphy, Ryan D.; Mallet, T. Conn; Crane, Edward J.
- Abstract
We have cloned NADH oxidase homologues fromPyrococcus horikoshiiandP. furiosus, and purified the recombinant form of theP. horikoshiienzyme to homogeneity fromEscherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+→2CoA-SH + NAD(P)+). TheP. horikoshiienzyme shows akcat app of 7.2 s−1 with NADPH at 75 °C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equalkcats, while theK m for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source.P. furiosuscells were assayed for small molecular mass thiols and found to contain 0.64 µmol CoA·g dry weight−1 (corresponding to 210 µmCoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.
- Subjects
COENZYMES; NAD(P)H dehydrogenases; ENZYMES; PROTEINS; ARCHAEBACTERIA; ESCHERICHIA coli
- Publication
FEBS Journal, 2005, Vol 272, Issue 5, p1189
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2005.04555.x