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Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii
Author(s) -
Harris Dennis R.,
Ward Donald E.,
Feasel Jeremy M.,
Lancaster Kyle M.,
Murphy Ryan D.,
Mallet T. Conn,
Crane Edward J.
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04555.x
Subject(s) - pyrococcus furiosus , pyrococcus horikoshii , nad+ kinase , cofactor , biochemistry , enzyme , chemistry , hyperthermophile , reductase , stereochemistry , archaea , gene
We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus , and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia 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) + ). The P. horikoshii enzyme shows a k cat 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 equal k cat s, while the K 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 EH 2 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. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 µmol CoA·g dry weight −1 (corresponding to 210 µ m CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.