title: Oxidized NADH oxidase inhibits activity of an ATP/NAD kinase from a Thermophilic archaeon.
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.
authors.affiliation: College of Plant Sciences, Jilin University, Changchun 130-062, China.
authors.forename: Baolei
authors.initials: B
authors.lastname: Jia
authors.forename: Sangmin
authors.initials: S
authors.lastname: Lee
authors.forename: Bang Phuong
authors.initials: BP
authors.lastname: Pham
authors.forename: Jinliang
authors.initials: J
authors.lastname: Liu
authors.forename: Hongyu
authors.initials: H
authors.lastname: Pan
authors.forename: Shihong
authors.initials: S
authors.lastname: Zhang
authors.forename: Gang-Won
authors.initials: GW
authors.lastname: Cheong
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000008:administration & dosage / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D016415:Sequence Alignment; D019712:Thermococcus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); D010100:Oxygen