Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii

Abstract

While surveying the genomes of hyperthermophilic and thermophilic Archaea for homologues of the flavoprotein disulfide reductases, many homologues with a high degree of identity to the branch of this family represented by glutathione reductase were found [1]. Most of the homologues appear to belong to the subfamily that depend on a redox-active single cysteine, analogous to the NADH oxidase and per-oxidase of Enterococcus and the coenzyme A disulfide reductase (CoADR; EC 1.8.1.14) of Staphylococcus Correspondence E. J. Crane III, 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 + fi 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 dephos-pho-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. furio-sus cells were assayed for small molecular mass thiols and found to contain 0.64 lmol CoAAEg dry weight)1 (corresponding to 210 lm CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents. Abbreviations CoADR, coenzyme A disulfide reductase (EC# 1.8.1.14); pfCoADR, P. furiosus coenzyme A disulfide reductase; phCoADR, P. horikoshii coenzyme A disulfide reductase; DTNB, 5,5¢ dithiobis(2-nitrobenzoic acid); EH 2 , two-electron reduced enzyme; EH 4 , four-electron reduced enzyme; HEPPS, N-(2-hydroxyethyl)piperazine-N¢-3-propanesulfonic acid; NOX, NADH oxidase; NPX, NADH peroxidase; TCA, trichloroacetic acid.

Structure of Coenzyme A−Disulfide Reductase from Staphylococcus aureus at 1.54 Å Resolution,

“…GSH, on the other hand, is not produced by the Archaea; it is also absent in many Bacteria. Harris et al (58) recently reported that CoASH is the major low-molecular weight thiol in the hyperthermophilic archaeon Pyrococcus furiosus, and the recombinant form of a CoADR from Pyrococcus horikoshii has been characterized. The enzyme exhibits a preference for NADPH in the reduction of CoAD, but questions remain regarding both the identity of the nonflavin redox center and the high NAD(P)H oxidase activity observed in the presence of FAD.…”

Characterization of NADH Oxidase/NADPH Polysulfide Oxidoreductase and Its Unexpected Participation in Oxygen Sensitivity in an Anaerobic Hyperthermophilic Archaeon

“…The tk1481 gene consisted of 1,344 bp and encoded a protein of 448 amino acids with a predicted molecular mass of 48,780 Da. The deduced amino acid sequence shared 24 to 33% identity with the other six NOX paralogs in T. kodakarensis and showed 31 to 35% identity with previously reported NOXs from hyperthermophiles, including H 2 O 2 -H 2 O-forming NOX1 from P. furiosus (PF1532) (33%) (38), H 2 O 2 -forming NoxA-2 from A. fulgidus (AF0395) (32%) (28), H 2 O-forming NOX from Thermococcus profundus (31%) (14), CoADR from P. horikoshii (PH0572) (31%) (11), and NAD(P)H:elemental sulfur oxidoreductase from P. furiosus (PF1186) (35%) (34). The homology of TK1481 with well-studied NOXs from mesophiles was lower, as demonstrated by the 27 to 28% identity with H 2 O-forming NOXs from L. sanfranciscensis (18), Streptococcus mutans (12), and Enterococcus faecalis (29) and the 25% identity with H 2 O 2 -forming NOX from S. mutans (12).…”

Characterization of an NADH oxidase of the flavin-dependent disulfide reductase family from Methanocaldococcus jannaschii

“…In an NADP-utilizing enzyme the C-terminal Gly of this motif is replaced with Ala, Pro or Ser, perhaps to accommodate the pyrophosphate group of NADP (Bellamacina, 1996). It should be noted that CoADRs from Bacillus anthracis and P. horikoshii carry the GXGXXG motif, yet oxidize both NADH and NADPH (Harris et al, 2005;Wallen et al, 2008).…”

“…Some of these non-methanogenic archaea might use Nox homologues to counter the toxic effect of the molecular oxygen that they could be exposed to in their natural habitats (Kengen et al, 2003;Ward et al, 2001). Also, in Pyrococcus horikoshii and Pyrococcus furiosus, a Nox homologue has been demonstrated to act as a coenzyme A disulfide reductase (CoADR) (Harris et al, 2005) as well as sulfur reductase (Schut et al, 2007). None of the methanogen Nox homologues have been investigated for their catalytic or in vivo functions.…”