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Oxidized NADH Oxidase Inhibits Activity of an ATP/NAD Kinase from a Thermophilic Archaeon

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Abstract

NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization. Under physiological conditions, unoxidized and oxidized NOXtk formed dimers and hexamers, respectively. Mutating the single cysteine residue Cys45 to alanine (NOXtkC45A) decreased NADH oxidase activity without affecting dimerization or hexamerization, suggesting that oligomerization does not occur through disulfide bond formation. Pull-down assay results indicated that an ATP/NAD kinase from T. kodakarensis KOD1 (ANKtk) binds to NOXtk. Use of several assays revealed that ANKtk can only bind to oxidized hexameric NOXtk, through which it inhibits ANKtk activity. Because ANKtk converts NADH to NADPH (an important factor in oxidative stress protection), a model based on in vitro result was proposed in which NOXtk hexamerization under oxic conditions inhibits both NOXtk and ANKtk activities, thereby sensitizing cells to oxidative stress-induced death.

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Abbreviations

NOX:

NADH oxidase

ANK:

ATP/NAD kinase

native-PAGE:

Native polyacrylamide gel electrophoresis

CBB:

Coomassie Brilliant Blue

PVDF:

Poly(vinylidene difluoride)

SDS–PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. College of Plant Sciences, Jilin University, Changchun, 130-062, China

    Baolei Jia, Jinliang Liu, Hongyu Pan & Shihong Zhang

  2. Division of Applied Life Sciences (BK21 program), Gyeongsang National University, Jinju, 660-701, Korea

    Baolei Jia, Sangmin Lee, Bang Phuong Pham & Gang-Won Cheong

  3. Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, 660-701, Korea

    Gang-Won Cheong

Authors
  1. Baolei Jia
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  2. Sangmin Lee
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  3. Bang Phuong Pham
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  4. Jinliang Liu
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  5. Hongyu Pan
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  6. Shihong Zhang
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  7. Gang-Won Cheong
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Corresponding author

Correspondence to Gang-Won Cheong.

Additional information

B. Jia and S. Lee contributed equally to the paper.

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Jia, B., Lee, S., Pham, B.P. et al. Oxidized NADH Oxidase Inhibits Activity of an ATP/NAD Kinase from a Thermophilic Archaeon. Protein J 29, 609–616 (2010). https://doi.org/10.1007/s10930-010-9284-y

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  • DOI: https://doi.org/10.1007/s10930-010-9284-y

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