Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii. Implications for this disulfide metabolism of anaerobic hyperthermophiles.
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 kcat app of 7.2 s(-1) with NADPH at 75 degrees C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km 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. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 micromol CoA.g dry weight(-1) (corresponding to 210 microM CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.
Harris
Dennis R
DR
Genencor International, Palo Alto, CA, USA.
Ward
Donald E
DE
Feasel
Jeremy M
JM
Lancaster
Kyle M
KM
Murphy
Ryan D
RD
Mallet
T Conn
TC
Crane
Edward J
EJ
D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000378:metabolism*; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism
D019641:DNA, Archaeal; D013438:Sulfhydryl Compounds; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C017145:coenzyme A disulfide; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C014703:disulfide reductase (NADH); D003065:Coenzyme A