Publications

• Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum.

  Hillmann F., Riebe O., Fischer R.J., Mot A., Caranto J.D., Kurtz D.M. Jr., Bahl H.

  Two flavo-diiron proteins (FDPs), FprA1 and FprA2, are up-regulated when the strictly anaerobic solvent producer, Clostridium acetobutylicum, is exposed to dioxygen. These two FDPs were purified following heterologous overexpression in Escherichia coli as N-terminal Strep-tag fusion proteins. The ... >> More

  Two flavo-diiron proteins (FDPs), FprA1 and FprA2, are up-regulated when the strictly anaerobic solvent producer, Clostridium acetobutylicum, is exposed to dioxygen. These two FDPs were purified following heterologous overexpression in Escherichia coli as N-terminal Strep-tag fusion proteins. The recombinant FprA1 and FprA2 were found to be homodimeric and homotetrameric, respectively, and both FDPs functioned as terminal components of NADH oxidases (NADH:O(2) oxidoreductases) when using C. acetobutylicum NADH:rubredoxin oxidoreductase (NROR) and rubredoxin (Rd) as electron transport intermediaries. Both FDPs catalyzed the four-electron reduction of molecular oxygen to water with similar specific activities. The results are consistent with these FDPs functioning as efficient scavengers of intracellular dioxygen under aerobic or microoxic growth conditions. << Less

  FEBS Lett. 583:241-245(2009) [PubMed] [EuropePMC]

• Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria.

  Kawasaki S., Ishikura J., Chiba D., Nishino T., Niimura Y.

  Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O(2). When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O(2)-free N(2) carrier-gas) to microoxic (sparg ... >> More

  Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O(2). When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O(2)-free N(2) carrier-gas) to microoxic (sparged with 3% O(2)/97% N(2) mixed carrier-gas) growth conditions in the mid exponential phase (OD(660)=1.0). When the strain grew under 3% O(2)/97% N(2), the medium remains anoxic. Thirty minutes after beginning aeration with 3% O(2), the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration. We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe. The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts. The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa. The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor. The final product of NADH oxidation was H(2)O, and the estimated K(m) for oxygen was 61.9 microM. These data demonstrate that an O(2)-response enzyme that is capable of detoxifying oxygen to water exists in C. aminovalericum. << Less

  Arch Microbiol 181:324-330(2004) [PubMed] [EuropePMC]

• O2 and reactive oxygen species detoxification complex, composed of O2-responsive NADH:rubredoxin oxidoreductase-flavoprotein A2-desulfoferrodoxin operon enzymes, rubperoxin, and rubredoxin, in Clostridium acetobutylicum.

  Kawasaki S., Sakai Y., Takahashi T., Suzuki I., Niimura Y.

  Clostridium acetobutylicum, an obligate anaerobe, grows normally under continuous-O(2)-flow culture conditions, where the cells consume O(2) proficiently. An O(2)-responsive NADH:rubredoxin oxidoreductase operon composed of three genes (nror, fprA2, and dsr), encoding NROR, functionally uncharacte ... >> More

  Clostridium acetobutylicum, an obligate anaerobe, grows normally under continuous-O(2)-flow culture conditions, where the cells consume O(2) proficiently. An O(2)-responsive NADH:rubredoxin oxidoreductase operon composed of three genes (nror, fprA2, and dsr), encoding NROR, functionally uncharacterized flavoprotein A2 (FprA2), and the predicted superoxide reductase desulfoferrodoxin (Dsr), has been proposed to participate in defense against O(2) stress. To functionally characterize these proteins, native NROR from C. acetobutylicum, recombinant NROR (rNROR), FprA2, Dsr, and rubredoxin (Rd) expressed in Escherichia coli were purified. Purified native NROR and rNROR both exhibited weak H(2)O(2)-forming NADH oxidase activity that was slightly activated by Rd. A mixture of NROR, Rd, and FprA2 functions as an efficient H(2)O-forming NADH oxidase with a high affinity for O(2) (the K(m) for O(2) is 2.9 +/-0.4 microM). A mixture of NROR, Rd, and Dsr functions as an NADH-dependent O(2)(-) reductase. A mixture of NROR, Rd, and rubperoxin (Rpr, a rubrerythrin homologue) functions as an inefficient H(2)O-forming NADH oxidase but an efficient NADH peroxidase with a low affinity for O(2) and a high affinity for H(2)O(2) (the K(m)s for O(2) and H(2)O(2) are 303 +/-39 microM and <or=1 microM, respectively). A gene encoding Rd is dicistronically transcribed with a gene encoding a glutaredoxin (Gd) homologue, and the expression levels of the genes encoding Gd and Rd were highly upregulated upon exposure to O(2). Therefore, nror operon enzymes, together with Rpr, efficiently function to scavenge O(2), O(2)(-), and H(2)O(2) by using an O(2)-responsive rubredoxin as a common electron carrier protein. << Less

  Appl. Environ. Microbiol. 75:1021-1029(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H2O.

  Jia B., Park S.C., Lee S., Pham B.P., Yu R., Le T.L., Han S.W., Yang J.K., Choi M.S., Baumeister W., Cheong G.W.

  An NADH oxidase (NOX) was cloned from the genome of Thermococcus profundus (NOXtp) by genome walking, and the encoded protein was purified to homogeneity after expression in Escherichia coli. Subsequent analyses showed that it is an FAD-containing protein with a subunit molecular mass of 49 kDa th ... >> More

  An NADH oxidase (NOX) was cloned from the genome of Thermococcus profundus (NOXtp) by genome walking, and the encoded protein was purified to homogeneity after expression in Escherichia coli. Subsequent analyses showed that it is an FAD-containing protein with a subunit molecular mass of 49 kDa that exists as a hexamer with a native molecular mass of 300 kDa. A ring-shaped hexameric form was revealed by electron microscopic and image processing analyses. NOXtp catalyzed the oxidization of NADH and NADPH and predominantly converted O(2) to H(2)O, but not to H(2)O(2), as in the case of most other NOX enzymes. To our knowledge, this is the first example of a NOX that can produce H(2)O predominantly in a thermophilic organism. As an enzyme with two cysteine residues, NOXtp contains a cysteinyl redox center at Cys45 in addition to FAD. Mutant analysis suggests that Cys45 in NOXtp plays a key role in the four-electron reduction of O(2) to H(2)O, but not in the two-electron reduction of O(2) to H(2)O(2). << Less

  FEBS J 275:5355-5366(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus.

  Zhang Y.W., Tiwari M.K., Gao H., Dhiman S.S., Jeya M., Lee J.K.

  NADH oxidase (Nox) catalyzes the conversion of NADH to NAD(+). A previously uncharacterized Nox gene (LrNox) was cloned from Lactobacillus rhamnosus and overexpressed in Escherichia coli BL21(DE3). Sequence analysis revealed an open reading frame of 1359 bp, capable of encoding a polypeptide of 45 ... >> More

  NADH oxidase (Nox) catalyzes the conversion of NADH to NAD(+). A previously uncharacterized Nox gene (LrNox) was cloned from Lactobacillus rhamnosus and overexpressed in Escherichia coli BL21(DE3). Sequence analysis revealed an open reading frame of 1359 bp, capable of encoding a polypeptide of 453 amino acid residues. The molecular mass of the purified LrNox enzyme was estimated to be ~50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 100 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had optimal activity at pH 5.6 and temperature 65 °C, and k(cat)/K(m) of 3.77×10(7) s(-1) M(-1), the highest ever reported. Heat inactivation studies revealed that LrNox had high thermostability, with a half-life of 120 min at 80 °C. Molecular dynamics simulation studies shed light on the factors contributing to the high activity of LrNox. Although the properties of Nox from several microorganisms have been reported, this is the first report on the characterization of a recombinant H(2)O-forming Nox with high activity and thermostability. The characteristics of the LrNox enzyme could prove to be of interest in industrial applications such as NAD(+) regeneration. << Less

  Enzyme Microb Technol 50:255-262(2012) [PubMed] [EuropePMC]

• Identification of two distinct NADH oxidases corresponding to H2O2-forming oxidase and H2O-forming oxidase induced in Streptococcus mutans.

  Higuchi M., Shimada M., Yamamoto Y., Hayashi T., Koga T., Kamio Y.

  Two distinct NADH oxidases, corresponding to H2O2-forming and H2O-forming enzymes were purified to homogeneity from Streptococcus mutans and their basic properties determined. The H2O2-forming enzyme was a tetramer with a subunit molecular mass of about 56 kDa and required flavin adenine dinucleot ... >> More

  Two distinct NADH oxidases, corresponding to H2O2-forming and H2O-forming enzymes were purified to homogeneity from Streptococcus mutans and their basic properties determined. The H2O2-forming enzyme was a tetramer with a subunit molecular mass of about 56 kDa and required flavin adenine dinucleotide (FAD) for full activity. The enzyme had an isoelectric point of 6.6 and exhibited optimal activity at pH 6.0. The H2O-forming enzyme was a monomer with a molecular mass of 50 kDa and activity independent of exogenously added flavin. The enzyme had an isoelectric point of 4.8 and exhibited optimal activity between pH 7.0 and 7.5. Both enzymes oxidized NADH (Km 0.05 and 0.025 mM for the H2O2- and H2O-forming enzyme, respectively) but not NADPH and contained 1 mol of FAD per monomer. Spectra of the oxidized enzymes exhibited maxima at 271, 383 and 449 nm for the H2O2-forming enzyme and 271, 375 and 447 nm for the H2O-forming enzyme. Antibodies raised against the H2O2-forming enzyme or the H2O-forming enzyme reacted with their corresponding antigen, but did not cross-react. The amino-terminal regions of the two enzymes had completely different amino acid sequences. << Less

  J Gen Microbiol 139:2343-2351(1993) [PubMed] [EuropePMC]

• An archaeal NADH oxidase causes damage to both proteins and nucleic acids under oxidative stress.

  Jia B., Lee S., Pham B.P., Cho Y.S., Yang J.K., Byeon H.S., Kim J.C., Cheong G.W.

  NADH oxidases (NOXs) catalyze the two-electron reduction of oxygen to H2O2 or four-electron reduction of oxygen to H2O. In this report, we show that an NADH oxidase from Thermococcus profundus (NOXtp) displays two forms: a native dimeric protein under physiological conditions and an oxidized hexam ... >> More

  NADH oxidases (NOXs) catalyze the two-electron reduction of oxygen to H2O2 or four-electron reduction of oxygen to H2O. In this report, we show that an NADH oxidase from Thermococcus profundus (NOXtp) displays two forms: a native dimeric protein under physiological conditions and an oxidized hexameric form under oxidative stress. Native NOXtp displays high NADH oxidase activity, and oxidized NOXtp can accelerate the aggregation of partially unfolded proteins. The aggregates formed by NOXtp have characteristics similar to beta-amyloid and Lewy bodies in neurodegenerative diseases, including an increase of beta-sheet content. Oxidized NOXtp can also bind nucleic acids and cause their degradation by oxidizing NADH to produce H2O2. Furthermore, Escherichia coli cells expressing NOXtp are less viable than cells not expressing NOXtp after treatment with H2O2. As NOXtp shares similar features with eukaryotic cell death isozymes and life may have originated from hyperthermophiles, we suggest that NOXtp may be an ancestor of cell death proteins. << Less

  Mol Cells 29:363-371(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Molecular cloning and sequence analysis of the gene encoding the H2O-forming NADH oxidase from Streptococcus mutans.

  Matsumoto J., Higuchi M., Shimada M., Yamamoto Y., Kamio Y.

  To discover the molecular properties of two distinct NADH oxidases, corresponding to H2O2-forming oxidase (NOX-1) and H2O-forming oxidase (NOX-2) induced in Streptococcus mutans, for the first step we had cloned and sequenced the nox-1 gene encoding NOX-1. In this paper, a nox-2 gene encoding NOX- ... >> More

  To discover the molecular properties of two distinct NADH oxidases, corresponding to H2O2-forming oxidase (NOX-1) and H2O-forming oxidase (NOX-2) induced in Streptococcus mutans, for the first step we had cloned and sequenced the nox-1 gene encoding NOX-1. In this paper, a nox-2 gene encoding NOX-2 from S. mutans was cloned, and the nucleotides sequenced. The nox-2 gene comprises 1371 base-pairs, encoding a polypeptide of 457 amino acid residues. The deduced relative molecular mass (M(r) = 49919) agreed with the previous value obtained from the purified NOX-2 protein. The nox-2 gene was expressed in Escherichia coli using its own promoter. Alignment of the NOX-2 protein sequence with that of the NOX-1 showed that the proteins do not significantly resemble each other. Comparisons with the NADH oxidase from Streptococcus faecalis 10C1 yield identities of 41%. The redox-active cysteine in the enzyme from S. faecalis was found to correspond to Cys 44 in the NOX-2. << Less

  Biosci Biotechnol Biochem 60:39-43(1996) [PubMed] [EuropePMC]

• Isolation and properties of an H2O-forming NADH oxidase from Streptococcus faecalis.

  Schmidt H.L., Stocklein W., Danzer J., Kirch P., Limbach B.

  An H2O-forming NADH oxidase from Streptococcus faecalis, recently described [Hoskins, D. D., Whiteley, H. R. and Mackler, B. (1962) J. Biol. Chem. 237, 2647-2651], has been isolated as a uniform protein with specific activity 690 U/mg in a total yield of 50% by a two-step affinity chromatography p ... >> More

  An H2O-forming NADH oxidase from Streptococcus faecalis, recently described [Hoskins, D. D., Whiteley, H. R. and Mackler, B. (1962) J. Biol. Chem. 237, 2647-2651], has been isolated as a uniform protein with specific activity 690 U/mg in a total yield of 50% by a two-step affinity chromatography procedure. The enzyme is metal-free and has a molecular mass of about 51 000 Da and probably consists of a single polypeptide chain. As shown by fluorimetric titration, the prosthetic group is 1 mol FAD/mol protein. The affinity behaviour of the enzyme gives evidence for the existence of a dinucleotide-binding domain capable of binding NADH or FAD. The enzyme is specific for NADH (Km = 4.1 X 10(-5) M), NADPH is not oxidized. O2 is the preferred electron acceptor, in addition FAD and, very slowly, one-electron acceptors are reduced. It is not clear whether the reduction of FAD proceeds through the dinucleotide-binding site or by exchange of the prosthetic group. The stoichiometry of the reaction with O2 corresponds to the consumption of 2 mol NADH/mol O2, and only H2O is formed (2 NADH + 2 H+ + O2----2 NAD+ + 2 H2O). Neither H2O2 nor O2.-is detected as intermediate and H2O2 cannot replace O2 as an oxidant. The enzyme can, mainly in its reduced state, be inhibited by -SH reagents. Spectral data give no evidence for the existence of radical intermediates during reduction. The enzyme can obviously accept more than two electrons/mol. On the basis of these data two possible reaction mechanisms are discussed. A proposal for the biological purpose of the reaction is made. << Less

  Eur J Biochem 156:149-155(1986) [PubMed] [EuropePMC]