TK1299, a highly thermostable NAD(P)H oxidase from kodakaraensis exhibiting higher enzymatic activity with NADPH

Muhammad Atif Nisar,1 Naeem Rashid,1,* Qamar Bashir,1 Qurra-tul-Ann Afza Gardner,1 Muhammad Hassan Shafiq,1 and Muhammad Akhtar1,2

School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Punjab, Pakistan1 and School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK2

Received 27 November 2012; accepted 28 January 2013 Available online 26 February 2013

Seven nicotinamide adenine dinucleotide oxidase homologs have been found in the genome of Thermococcus koda- karaensis. The gene encoding one of them, TK1299, consisted of 1326 nucleotides, corresponding to a polypeptide of 442 amino acids. To examine the molecular properties of TK1299, the structural gene was cloned, expressed in Escherichia coli and the gene product was characterized. Molecular weight of the recombinant protein was 49,375 Da when determined by matrix-assisted laser desorption/ionization time-of-flight and 300 kDa when analyzed by gel filtration chromatography indicating that it existed in a hexameric form. The enzyme was highly thermostable even in boiling water where it exhibited more than 95% of the enzyme activity after incubation of 150 min. TK1299 catalyzed the oxidation of NADH as well as NADPH and predominantly converted O2 to H2O (more than 75%). Km value of the enzyme towards NADH and NADPH was almost same (24 ± 2 mM) where as specific activity was higher with NADPH compared to NADH. To our knowledge this is the most thermostable and unique NAD(P)H oxidase displaying higher enzyme activity with NADPH. Ó 2013, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Hyperthermophile; Thermococcus kodakaraensis; NAD(P)H oxidase; Cloning; Circular dichroism; Thermostable]

Nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a model hyperthermophilic organism whose whole genome has oxidases (NOXs, E.C.1.6.99.3) are members of “two dinucleotide been sequenced and reported (5). We have previously characterized binding domains” flavoproteins (tDBDF) superfamily. They catalyze two of the NADH oxidases, TK0304 (6) and TK1392 (7), out of seven the oxidation of NAD(P)H by reducing molecular oxygen to either present in T. kodakaraensis genome (8). In the present study we hydrogen peroxide (a two-electron reduction) or water (a four- focus on another NOX homolog, TK1299, from T. kodakaraensis. The electron reduction) or both (1). NOXs catalyze diverse redox reac- recombinant protein, produced in Escherichia coli, was purified and tions which are initiated by a stereospecific hydride transfer characterized in terms of its ability to reduce oxygen. between two cofactors, a pyridine nucleotide and a flavin adenine dinucleotide (FAD) (2). NOX homologs are widely distributed in MATERIALS AND METHODS members of all the three domains of life including eucarya, bacteria and . Genomes analyses have revealed the presence of Bacterial strains, plasmids, and media E. coli DH5a cells were used for variety of NOX homologs in anaerobic archaea, a class of organisms cloning purposes and BL21-CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA, USA) cells that have not been expected to possess NADH oxidases, indicating were employed for the expression of TK1299 gene that was cloned in pET-28a that they are well equipped for responding to oxidative stress. (Novagen, Madison, WI, USA). E. coli transformants were cultivated at 37 Cin e fi m Among anaerobic archaea the members of family Thermococcaceae Luria Bertani medium containing ampicillin at a nal concentration of 100 g/mL. contain multiple homologs of NOX (Table 1). Construction of the expression vector The structural gene, TK1299, fi Thermococcus kodakaraensis KOD1 is a hyperthermophilic encoding an NAD(P)H oxidase homolog was ampli ed by polymerase chain reaction (PCR) using genomic DNA of T. kodakaraensis as a template and a pair of primers archaeon isolated from a solfatara on the shore of Kodakara Island in consisting of TK1299-F (5-CCATGGAGAGAAAGACGGTCGTTG-3) and TK1299-R (5- Kagoshima, Japan (3,4). It is an obligate heterotroph, generally GTCAGAACTTGAGGACCCTGGC-3). Underlined sequence in TK1299-F indicates NcoI prefers proteinaceous matter as carbon and energy sources, and its recognition site. The amplified DNA fragment of 1.3 kbp was cloned in pTZ57R/T vector. The resulting plasmid was named pTZ-TK1299. TK1299 gene was liberated growth is associated with the reduction of elemental sulfur to H2S. It from pTZ-TK1299 using NcoI and BamHI restriction enzymes and cloned in pET-28a at the corresponding sites. The absence of any mutation in the cloned gene was confirmed by DNA sequencing. The resulting plasmid, pET-TK1299, was used to * Corresponding author. Tel.: þ92 42 99231534; fax: þ92 42 99230980. transform E. coli BL21-CodonPlus(DE3)-RIL for expression of TK1299. E-mail addresses: [email protected], [email protected] Expression and purification of recombinant TK1299 E. coli cells harboring (N. Rashid). pET-TK1299 were grown to early log phase and gene expression was induced for 4 h

Reproduced from J. Biosci. Bioeng. 116: 39-44 (2013).

Naeem Rashid: Participant of the 21st UM, 1993-1994.

353 TABLE 1. Distribution of NOX homologs in anaerobic archaea. temperatures ranging from 40Cto90C. Activation energy was calculated from straight line section of the Arrhenius plot. Thermodynamic parameters were Organism No. of homologs obtained by fitting the data to Eyring equation. Methanocaldococcus jannaschii 1 Circular dichroism analysis The structure stability of TK1299 was analyzed Methanococcus maripaludis 1 by circular dichroism (CD) spectroscopy in the presence or absence of the prosthetic Methanosarcina acetivorans 2 group (FAD). The protein samples were incubated at different temperatures ranging Methanosarcina barkeri 1 from 20Cto100C for 15 min in water bath or in autoclave for 5 min and CD spectra abyssi 4 were taken. To examine the protein stability in the presence of cofactor, enzyme 6 solutions were saturated with FAD and then incubated at different temperatures 5 before recording the CD spectra. Similarly the enzyme stability was also checked in Thermococcus gammatolerans 5 the presence of excess FAD. The CD spectra of the protein solutions were recorded in Thermococcus kodakaraensis 7 10 mM TriseCl pH 7.5 in the far UV-range of 200e260 nm. Solvent spectra were Thermococcus onnurineus 7 subtracted from those of the protein solutions. The spectra were recorded at Thermococcus sibiricus 5 Chirascan-plus CD Spectrometer (Applied Photophysics, UK).

at 37 C using 0.4 mM isopropyl-thio-b-D-galactoside (IPTG) in LB medium con- RESULTS taining ampicillin at a final concentration of 100 mg/mL. The cells were harvested by e centrifugation at 6000 g for 5 min at 4 C, resuspended in 50 mM Tris Cl (pH 8.0), Sequence analysis of TK1299 When multiple sequence and disrupted by sonication at 4C for 10 min. The supernatant after centrifugation was heated at 80C for 20 min. The heat labile proteins from the host cells were alignment of TK1299 was performed, using the amino acid denatured and removed by centrifugation at 13,000 g for 15 min at 4C. The sequence of its homologs, we could identify NAD(P)H binding heat-stable recombinant protein in the supernatant was further purified using motif, one coenzyme A disulfide reductase motif and two FAD fi ÄKTA Puri er chromatography system (GE Healthcare, Uppsala, Sweden). The binding motifs. Archaeal specific NADH oxidase amino acids (10) heat-treated cell extract was applied to anion-exchange Resource Q (6 mL) column (GE Healthcare) and recombinant TK1299 was eluted with a linear were also completely conserved in TK1299 (Fig. 1). TK1299 gradient of 0e1 M NaCl. The fractions containing TK1299 were dialyzed against displayed low homology (26e42%) to six NOX paralogs in 1.25 M (NH4)2SO4 in 50 mM sodium phosphate buffer (pH 7.0). The dialyzed T. kodakaraensis. enzyme sample was applied to hydrophobic Resource PHE (1 mL) column A phylogenetic tree was constructed by comparing the amino equilibrated with 1.25 M (NH4)2SO4 in 50 mM sodium phosphate buffer (pH 7.0) acid sequence of all the seven NADH oxidases found in and elution was done by decreasing the concentration of (NH4)2SO4. Analysis of the purified TK1299 was performed by sodium dodecyl sulfate polyacrylamide gel T. kodakaraensis genome and their homologs which resulted in electrophoresis (SDS-PAGE). seven discrete clusters (Fig. 2). Among the NADH oxidases that have Quantification of FAD bound to TK1299 To quantify FAD bound per mole of been characterized, TK1299 displayed highest homologies of 91% recombinant TK1299, 400 ml of TK1299 solution was mixed with 44 ml 50% tri- (identity) with that originated from chloroacetic acid (TCA). The mixture was held on ice for 5 min and then centrifuged (accession no. CAQ43117) (11), 88 with Pyrococcus furiosus NAD(P)H at 12,000 rpm for 10 min to pellet down Apo-TK1299. The pellet was resuspended in elemental sulfur oxidoreductase (accession no. AAL81310) (12), 83% 400 ml 5% TCA and recentrifuged to recover an additional supernatant that was fi combined with that obtained from the first centrifugation. FAD in this combined to Pyrococcus horikoshii CoA disul de reductase (accession no. supernatant was quantified by measuring the absorbance at 450 nm. Calculations NP_142538) (13), and 60% with Archaeoglobus fulgidus NADH were based on absorption coefficient value of 11.3 mM 1 cm 1 for FAD (9). Apo- oxidase (accession no. NP_069231) (14). TK1299 displayed a low e TK1299, in the pellet, was dissolved in 50 mM Tris Cl pH 8.0 containing 0.2% homology with well-studied bacterial NOXs, 28% with NAD(P)H sodium dodecyl sulfate (buffer A). Spectra of apo-TK1299 were taken using buffer A as blank. oxidase from Lactobacillus sanfranciscensis (15) (PDB code: 2CDU) Determination of molecular mass and quaternary structure of TK1299 The and 27% with NOX from Streptococcus pyogenes (16) (PDB code: subunit molecular mass of recombinant TK1299 was determined by SDS-PAGE. The 2BC0). Interestingly, TK1299 shared a significant high homology subunit molecular mass was also determined by matrix-assisted laser desorption/ (57%) with human apoptosis-inducing factor three (AIF-3; acces- ionization time-of-flight mass spectrometry (MALDI-TOF MS). The enzyme sample sion no. Q96NN9). The other six NADH oxidase homologs from (2 mg protein) was mixed with 20 ml of matrix-B (5 mg sinapinic acid dissolved in 1 mL T. kodakaraensis displayed a variable homology, ranging from 27% to of 30% acetonitrile containing 0.1% trifluoroacetic acid). From this sample mixture, 5 ml was spotted on stainless steel mass spectrometric plate and allowed to dry for 50%, with human apoptosis-inducing factor three. e fi 20 30 min. The mass spectrum of the puri ed enzyme was recorded by using Bruker Production and purification of TK1299 When production of Autoflex MALDI-TOF mass spectrophotometer (Bruker Daltonics Inc., MA, USA). Native molecular mass was determined by Superdex 200 10/300 GL gel filtration TK1299 in E. coli cells harboring pET-TK1299 was analyzed on SDS- column (GE Healthcare) equilibrated with 0.15 M NaCl in 50 mM phosphate buffer PAGE it was found that a high level of TK1299 was produced in the (pH 7.0). The calibration curve was obtained with the standard proteins including cells induced with IPTG and a leaky expression of TK1299 gene was ferritin (440 kDa), phosphorylase B (195 kDa), lactate dehydrogenase (140 kDa), present in the cells that were not induced. Soluble and insoluble malate dehydrogenase (70 kDa), and myoglobin (17 kDa). fractions, after lysis of the cells by sonication, were analyzed by SDS- Enzyme assay NAD(P)H oxidase activity of recombinant TK1299 was PAGE. The analysis showed that almost 90e95% of recombinant determined in a mixture containing 50 mM TriseCl buffer (pH 7.8), 220 mM NAD(P)H fi and appropriate amount of the enzyme. Normally the reaction mixture, without TK1299 was produced as soluble protein. The rst step of NAD(P)H, was kept for 5 min at respective temperature before the start of purification based on the thermostability of TK1299 was heat reaction. In most cases, prior to assays, the enzyme was incubated at room treatment which resulted in removal of most of the heat labile temperature with FAD. The oxidation of NAD(P)H was spectrophotometerically fi 1 1 E. coli proteins. TK1299 was further puri ed to apparent monitored as the decrease in absorbance at 340 nm ( 3 340 ¼ 6.22 mM cm ). One unit of NAD(P)H oxidase activity corresponds to the oxidation of 1 mmol of homogeneity by ion exchange and hydrophobic interaction NAD(P)H per min. chromatographies (Fig. 3). H2O2 detection Production of H2O2 was detected using Peroxi Detect Kit Molecular weight and quaternary structure of TK1299 The m (Sigma). The assay mixture containing 220 M NADH was incubated at 70 C until result of MALDI-TOF mass spectrometery analysis of recombinant the OD340 of solution was <0.01, indicating that almost all of the NAD(P)H had been oxidized. Then 100 ml of this solution was added to 1 mL of hydrogen peroxide color TK1299 showed that the molecular weight of the protein was reagent (ferrous ammonium sulfate dissolved in 2.5 M H2SO4) and incubated at 49375.032 Da (Fig. 4), while theoretically molecular weight room temperature for 30 min (until the color formation is completed). Under acidic calculated from the amino acid sequence of TK1299 was 2þ þ3 3þ conditions peroxide oxidizes Fe to Fe ions. Fe ions will then form colored 48609.98 Da. MALDI-TOF data indicated that FAD (molecular product with xylenol orange, which was observed both at 560 and 590 nm. The rates weight 785 Da) was tightly bound and flew together with TK1299 of spontaneous thermal degradation of NAD(P)H and H2O2 were determined by control experiments and were subtracted from the experimental reaction rates. giving a molecular weight of 49375.032 Da equivalent to the

Analyses of kinetic parameters Km and Vmax of recombinant TK1299 were holoenzyme. This result is consistent with the fact that NADH calculated by LineweavereBurk plot. Steady-state assays were performed at various oxidases are flavoproteins and contain bound-FAD molecules.

354 FIG. 1. Multiple sequence alignment of TK1299. Alignment was performed with ClustalW program provided at: http://www.ebi.ac.uk. FAD binding domains I and II are shown in boxes. NADH binding domain is overlined. Active site cysteine residue is shown by a star at the top. Archaeal specific NADH oxidase amino acids (10) are shown with filled circles at the top. Identical sequences are shown by asterisks at the bottom. The accession numbers are as follows: TK1299, YP_183712; T. profundus, CAQ43117; P. furiosus, NP_578915; P. horikoshii, NP_142538.

When we performed gel filtration chromatography to determine Upon removal of FAD, the absorption spectrum of apo-TK1299 did the subunit number, TK1299 eluted at a retention volume of not show any significant absorbance in the visible region 11.8 mL, suggesting the molecular mass of approximately 300 kDa. indicating the non-covalent attachment of FAD (Fig. 5B). When Keeping in view the subunit mass of 49.4 kDa, this result indicated we determined stoichiometry of TK1299 and FAD, we found that the enzyme exists in a hexameric form. 0.5 0.02 FAD attached per subunit of TK1299 lower than the Flavin content of the enzyme MALDI-TOF mass spec- previously reported NADH oxidases from hyperthermophiles trometery analysis indicated the presence of FAD in the purified (11,13) which contained 1 0.1 FAD per subunit. TK1299. The presence of flavin was further evidenced by Effect of temperature, pH and FAD on TK1299 activity We UVevisible spectrum. Purified enzyme had absorption maxima examined the enzyme activity of TK1299 at various temperatures approximately at 370 nm, with a shoulder at about 460 nm for 5 min and found that the activity of the enzyme increased with (Fig. 5A), which is a characteristic feature of flavoproteins (9). the increase in temperature till it reached highest at 75C(Fig. 6A).

355 TK1481 12 TON_0129 49375.032 TERMP_00352 TSIB_1897 TERMP_00960 10 PF1197 TSIB_1323 PF1532 TK0828 TK0304 TON_1271 8 PYCH_10130 TON_0865 PAB1931 6 TSIB_0263 PF1795 PH0572 PYCH_01670 4 PF1186 TSIB_0637 PYCH_07890 TON_0305 TERMP_00975 TK1299 2 TON_1265 TK0119 PAB0184 0 42500 45000 47500 50000 52500 55000 PAB1842 TK1392 TERMP_00228 Mass (Da) PF1245 TON_0204 PF2006 TK0116 PYCH_18340 fi PYCH_09850 FIG. 4. MALDI-TOF mass spectrum of the puri ed TK1299. The counts are shown on TON_1282 TSIB_0635 Y-axis and the deconvoluted mass in daltons on X-axis. Molecular mass of recombinant TK1299 is written at the top of the peak. 0.1

FIG. 2. Phylogenetic analysis of all the seven NADH oxidases found in T. kodakaraensis activity of the purified enzyme was measured without the addition and their homologs. The tree was constructed by ClustalW provided by DNA Data Bank of FAD. It was found that TK1299 exhibited an activity of 11 U/mg. In of Japan (http://clustalw.ddbj.nig.ac.jp/). Segments responding to an evolutionary the second experiment, the enzyme was first incubated with distance of 0.1 are shown. NAD(P)H homologs originating from T. kodakarensis are m shown in bold. Numbers at the termini correspond to the open reading frame anno- 220 M FAD and the enzyme activity was measured after passing tated on the respective genome. Abbreviations used are: PAB, ;PF, through PD-10 column to remove free FAD. In this case the enzyme Pyrococcus furiosus; PH, Pyrococcus horikoshii; PYCH, Pyrococcus yayanosii; TERMP, activity was enhanced two-times (25 U/mg) the activity obtained ; TK, Thermococcus kodakarensis; TON, Thermococcus onnur- without the addition of FAD. In the third experiment, the enzyme ineus; TSIB, Thermococcus sibiricus. was incubated in 220 mM FAD and enzyme activity was examined without removing the extra FAD. Under these conditions the Keeping the temperature constant at 75C, we examined the enzyme activity enhanced four-times (42 U/mg) the activity enzyme activity at various pH and found that TK1299 exhibited highest enzyme activity at pH 8.0 (Fig. 6B). We further examined the thermostability of TK1299 and found that it is highly thermostable with more than 95% residual activity even after A incubation of 150 min in boiling water. Another NADH oxidase, 0.15 Tk0304, from T. kodakaraensis is considered highly thermostable as it exhibited highest activity at 85e90C but it displayed a half- 0.14 life of 80 min in the boiling water (6). As TK1299 displayed no significance decrease in enzyme activity even after incubation of 0.13 150 min in the boiling water (Fig. 6C), we propose it the most thermostable NADH oxidase. 0.12 In order to examine the effect of FAD on TK1299 enzyme activity, Absorbance three different sets of experiments were performed. In the first set, 0.11

0.10 350 400 450 500 550 600 650 Wavelength (nm) B

0.13

0.12

0.11 Absorbance FIG. 3. Coomassie brilliant blue stained 12% SDS-PAGE demonstrating production and 0.10 step-wise purification of TK1299. Lane M, protein marker (no. SM0661, Fermentas); lane 1, total lysate of un-induced cells containing pET-TK1299 plasmid; lane 2, total 350 400 450 500 550 600 650 lysate of cells containing pET-TK1299 plasmid induced with 0.4 mM IPTG; lane 3, insoluble fraction of the sample in lane 2; lane 4, soluble fraction of the sample in lane Wavelength (nm) 2; lane 5, insoluble fraction, after heat treatment, of the sample in lane 4; lane 6, soluble fraction, after heat treatment, of the sample in lane 4; lane 7, sample after ion FIG. 5. UVevisible spectrum of purified TK1299. (A) Spectrum before (outset) and after exchange column; lane 8, sample after hydrophobic interaction chromatography. (inset) saturation with FAD. (B) Spectrum of apo-TK1299.

356 A 50

90 40

30 70

20 50 10

0 30 50 55 60 65 Relative activity (%) Temperature (˚C) 10 FIG. 7. Comparison of TK1299 activity at various temperatures using NADH and NADPH 40 50 60 70 80 90 as substrates. Filled and empty bars represent the enzyme activity with NADPH and NADH, respectively. The number of experiments for the data is 4. Temperature (˚C) B Arrhenius plot (ln k vs. 1/T), from which activation energy of 100 31.9 kJ mol 1 was calculated. Activation enthalpy (DH) and entropy (DS) were found to be 32 1 kJ mol 1 and 116 10 J mol 1 K 1 by 80 analyzing the data using Eyring plot. Negative value of activation entropy indicated the decrease in entropy upon achieving the transition state, reflecting an associative mechanism. 60 For examining the kinetic parameters various concentrations of NADH ranging from 0 to 880 mM were used and assays were 40 conducted at 75 C. The enzyme followed the MichaeliseMenten equation. The Vmax and Km values were found to be 83 1Umg 1 and 24 1 mM, respectively. The k value was found

Relative activity (%) cat 20 to be 68 1s 1. Similarly, when various concentrations of NADPH were used and assays were conducted at 65 C, Vmax, Km and kcat 1 m 1 0 values were 61 1Umg ,25 1 M and 50 1s respectively. If we analyze the enzyme activity of TK1299 at 65C, a 23 1% higher 6.0 7.0 8.0 9.0 enzyme activity was observed when NADPH was used as pH a substrate compared to NADH (Fig. 7). We also tried the enzyme C assay at higher than 65C but the results were not reliable because of the instability of NADPH at these temperatures. 100 Structure stability of TK1299 The structure stability of TK1299 was analyzed at different temperatures by CD spectros- copy. There was no significant change in the CD spectra up to 100C in the presence of FAD, indicating that the enzyme maintains its 80 secondary structure even at 100 C(Fig. 8). At 120 C the molecular ellipticity shifted near zero even in the presence of FAD indicating that the secondary structure of the enzyme is not stable at this 60 temperature. Estimation of the reaction product When we performed Relative activity (%) the estimation of the end product of the reaction catalyzed by TK1299 at 70 C, we found that approximately 25 2% H2O2 and 40 050100 150 200 20 Time (min) -3 10 FIG. 6. Effect of temperature and pH on TK1299 enzyme activity. (A) Optimal 10 ) x temperature of the enzyme activity. Assays were performed at various temperatures -1 20°C ranging from 40Cto80C under standard conditions. (B) Optimal pH for TK1299 0 50°C enzyme activity. Assays were conducted in universal buffer at 75 C. (C) Thermosta- 200 210 220 230 240 250 260 dmol bility of TK1299. TK1299 was incubated in the boiling water for various intervals of 2 -10 70°C time and residual activity was examined at 75C and pH 8.0. The number of experi- ments for the data is 4. 90°C -20 100°C (deg cm autoclaved obtained without the addition of FAD. We further examined the -30 effect of various concentrations of FAD and found that TK1299 Molecular ellipticity m -40 exhibited highest activity in the presence of 180 M or above FAD. Wavelength (nm) Steady-state kinetics and kinetic parameters of TK1299 Up to 70 C, the increase in NADH oxidase activity followed a linear FIG. 8. CD spectra of TK1299 at various temperatures ranging from 20Cto120C.

357 fi 75 2% H2O was produced when NADH was oxidized and 3. Morikawa, M., Izawa, Y., Rashid, N., Hoaki, T., and Imanaka, T.: Puri cation and characterization of a thermostable thiol protease from a newly isolated approximately 13 1% H2O2 and 87 1% H2O was produced when hyperthermophilic Pyrococcus sp., Appl. Environ. Microbiol., 60, 4559e4566 NADPH was oxidized by TK1299. These results are in contrast to (1994). NOX1 from P. furiosus which is reported to produce 77% H2O2 and 4. Atomi, H., Fukui, T., Kanai, T., Morikawa, M., and Imanaka, T.: Description of 23% H2O (17). Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1, Archaea, 1, 263e267 DISCUSSION (2004). 5. Fukui, T., Atomi, H., Kanai, T., Matsumi, R., Fujiwara, S., and Imanaka, T.: Complete genome sequence of the hyperthermophilic archaeon Thermococcus TK1299 exhibited more than 90% identity to NADH oxidase from kodakaraensis KOD1 and comparison with Pyrococcus genomes, Genome Res., e T. profundus (NOXtp; B2G3S1_THEPR) which, under normal physi- 15, 352 363 (2005). ological conditions, is present in dimeric form while upon oxidative 6. Rashid, N., Hameed, S., Siddiqui, M. A., and Haq, I.: Gene cloning and char- acterization of NADH oxidase from Thermococcus kodakaraensis, Afr. J. Bio- stress it becomes hexameric protein. Most importantly, oxidized technol., 10, 17916e17924 (2011). NOXtp accelerated the aggregation of partially unfolded proteins 7. Rashid, N., Akmal, S., and Akhtar, M.: Gene cloning and characterization of and the degradation of nucleic acids in vitro, therefore it was TK1392, an NADH oxidase from Thermococcus kodakaraensis with a distinct e proposed that NOXtp may act as an ancestral cell death protein (18). C-terminal domain, Turk. J. Biochem., 36, 107 115 (2011). Moreover, TK1299 displayed a 57% identity to human apoptosis- 8. Kobori, H., Ogino, M., Orita, I., Nakamura, S., Imanaka, T., and Fukui, T.: Characterization of NADH oxidase/NADPH polysulfide oxidoreductase and its inducing factor-3 protein which is involved in programmed cell unexpected participation in oxygen sensitivity in an anaerobic hyper- death. Similarly TK1299 exhibited 88% identity to PF1186 of thermophilic archaeon, J. Bacteriol., 192, 5192e5202 (2010). P. furiosus (12) and 83% to PH0572 of P. horikoshii (13), and these 9. Scrutton, N. S.: Identification of covalent flavoproteins and analysis of the enzymes are reported to act as coenzyme A disulfide reductase covalent link, Methods Mol. Biol., 131, 181e193 (1999). 10. Case, C. L., Rodriguez, J. R., and Mukhopadhyay, B.: Characterization of an (CoADR) in vivo. The role of NAD(P)H oxidases in hyper- NADH oxidase of the flavin-dependent disulfide reductase family from Meth- thermophiles is still not clear. Generally, NADH oxidases are anocaldococcus jannaschii, Microbiology, 155,69e79 (2009). thought to be involved in defense against oxidative stress in both 11. Jia, B., Park, S. C., Lee, S., Pham, B. P., Yu, R., Le, T. L., Han, S. W., Yang, J. K., aerobic and anaerobic organisms. Higher enzyme activity of Choi, M. S., Baumeister, W., and Cheong, G. W.: Hexameric ring structure of TK1299 at temperature (75C) lower than the optimal growth a thermophilic archaeon NADH oxidase that produces predominantly H2O, FEBS J., 275, 5355e5366 (2008). temperature (85 C) of T. kodakaraensis KOD1 suggests that TK1299 12. Schut, G. J., Bridger, S. L., and Adams, M. W. W.: Insights into the metabolism may have a role in oxidative stress conditions as the dissolved of elemental sulfur by the hyperthermophilic archaeon Pyrococcus furiosus: oxygen concentration is higher at low temperature. Presence of characterization of a coenzyme A-dependent NAD(P)H sulfur oxidoreductase, J. Bacteriol., 189, 4431e4441 (2007). H2O and H2O2 producing NAD(P)H oxidases in T. kodakaraensis reflects that removal of oxygen in the cells may be done in two 13. Harris, D. R., Ward, D. E., Feasel, J. M., Lancaster, K. M., Murphy, R. D., Mallet, T. C., and Crane, E. J.: Discovery and characterization of a Coenzyme A fi steps: rstly O2 is converted into H2O and H2O2 with the help of disulfide reductase from Pyrococcus horikoshii. Implications for this disulfide NAD(P)H oxidases. If H2O2 is not removed rapidly, it may produce metabolism of anaerobic hyperthermophiles, FEBS J., 272, 1189e1200 (2005). hydroxyl radicals, which can be harmful to the cell. Therefore, in 14. Kengen, S. W., van der Oost, J., and de Vos, W. M.: Molecular characterization of H2O2-forming NADH oxidases from Archaeoglobus fulgidus, Eur. J. Biochem., second step H2O2 is removed or converted into H2O enzymatically 270, 2885e2894 (2003). utilizing NOXs, rubrerythrin or alkyl hydroxyperoxide reductase 15. Lountos, G. T., Jiang, R., Wellborn, W. B., Thaler, T. L., Bommarius, A. S., and (genes encoding these enzymes are found in T. kodakaraensis Orville, A. M.: The crystal structure of NAD(P)H oxidase from Lactobacillus genome), or chemically by reacting with pyruvate as reported in sanfranciscensis: insights into the conversion of O2 into two water molecules by Lactococcus lactis (1). However, detailed studies including gene the flavoenzyme, Biochemistry, 45, 9648e9659 (2006). disruption are needed in order to elucidate the role of TK1299 in 16. Seki, M., Iida, K., Saito, M., Nakayama, H., and Yoshida, S.: Hydrogen peroxide production in Streptococcus pyogenes: involvement of lactate oxidase and T. kodakaraensis cells. coupling with aerobic utilization of lactate, J. Bacteriol., 186, 2046e2051 (2004). References 17. Ward, D. E., Donnelly, C. J., Mullendore, M. E., van der Oost, J., de Vos, W. M., and Crane, E. J.: The NADH oxidase from Pyrococcus furiosus. Implications for 1. Miyoshi, A., Rochat, T., Gratadoux, J., Le, L. Y., Oliveira, S., and Azevedo, V.: the protection of anaerobic hyperthermophiles against oxidative stress, Eur. J. Oxidative stress in Lactococcus lactis, Genet. Mol. Res., 2, 348e358 (2003). Biochem., 268, 5816e5823 (2001). 2. Ojha, S., Meng, E. C., and Babbitt, P. C.: Evolution of function in the “two dinu- 18. Jia, B., Lee, S., Pham, B. P., Cho, Y. S., Yang, J. K., Byeon, H. S., Kim, J. K., and cleotide binding domains” flavoproteins, PLoS Comput. Biol., 3,1268e1280 Cheong, G. W.: An archaeal NADH oxidase causes damage to both proteins and (2007). nucleic acids under oxidative stress, Mol. Cells, 29, 363e371 (2010).

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