Báo cáo khoa học: Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H2O pot

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Hexameric ring structure of a thermophilic archaeonNADH oxidase that produces predominantly H2OBaolei Jia1,*, Seong-Cheol Park1,*,, Sangmin Lee1, Bang P. Pham1, Rui Yu1, Thuy L. Le1, SangWoo Han2,3, Jae-Kyung Yang4, Myung-Suk Choi4, Wolfgang Baumeister5and Gang-Won Cheong1,31 Division of Applied Life Sciences (BK21 Program), Gyeongsang National University, Jinju, Korea2 Department of Chemistry, Research Institute of Natural Science, Gyeongsang National University, Jinju, Korea3 Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Korea4 Division of Environmental Forest Science and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea5 Department of Molecular Structural Biology, Max-Planck-Institute for Biochemistry, Martinsried, GermanyThermococcus profundus is a thermophilic anaerobicarchaeon belonging to the Thermococcaceae familythat also includes Thermococcus kodakaraensis KOD1,a model thermophilic organism whose whole genomesequence has been reported [1]. As an anaerobe livingin deep-vent environments, it seems likely that Thermo-coccus encounters high levels of oxygen stress in thewater surrounding the vent [2]. In anaerobes, flavin-dependent NAD(P)H oxidases play an important roleprotecting organisms from oxidative stress [3].NADH oxidase (NOX) is a member of the flavo-protein disulfide reductase family that catalyzes thepyridine-nucleotide-dependent reduction of varioussubstrates, including O2,H2O2and thioredoxin [4].There are two types of NOX: those that catalyze thetwo-electron reduction of O2to H2O2and those thatcatalyze the four-electron reduction of O2to H2O [5].The physiological role of NOX is diverse, dependingon its substrates and products in different organisms.In anaerobic mesophiles, NOX enzymes, such as thoseKeywordselectron microscopy; H2O-producing;hexameric ring structure; NADH oxidase;thermophilic archaeonCorrespondenceG W. Cheong, Division of Applied LifeSciences, Gyeongsang National University,Jinju 660-701, KoreaFax: +82 55 752 7062Tel: +82 55 751 5962E-mail: gwcheong@gnu.ac.krPresent addressResearch Center for Proteineous Materials,Chosun University, Kwangju 501-759, Korea*These authors contributed equally to thiswork(Received 7 August 2008, revised 31 August2008, accepted 3 September 2008)doi:10.1111/j.1742-4658.2008.06665.xAn NADH oxidase (NOX) was cloned from the genome of Thermococ-cus profundus (NOXtp) by genome walking, and the encoded protein waspurified to homogeneity after expression in Escherichia coli. Subsequentanalyses showed that it is an FAD-containing protein with a subunitmolecular mass of 49 kDa that exists as a hexamer with a native molecularmass of 300 kDa. A ring-shaped hexameric form was revealed by electronmicroscopic and image processing analyses. NOXtp catalyzed the oxidiza-tion of NADH and NADPH and predominantly converted O2to H2O, butnot to H2O2, as in the case of most other NOX enzymess. To our knowl-edge, this is the first example of a NOX that can produce H2O predomi-nantly in a thermophilic organism. As an enzyme with two cysteineresidues, NOXtp contains a cysteinyl redox center at Cys45 in addition toFAD. Mutant analysis suggests that Cys45 in NOXtp plays a key role inthe four-electron reduction of O2to H2O, but not in the two-electronreduction of O2to H2O2.AbbreviationsCoADR, coenzyme A disulfide reductase; GR, glutathione reductase; Nbs2, 5,5¢-dithiobis-(2-nitrobenzoic acid); NOX, NADH oxidase(EC 1.6.99.3); NOXtp, Thermococcus profundus NADH oxidase.FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5355of Clostridium aminovalericum [6], Enterococcus (Strep-tococcus) and Lactococcus [3], are considered to beimportant enzymes in protecting against oxidativestress and in regenerating oxidized pyridine nucleo-tides through their capacity to reduce O2to H2Owithout the formation of harmful reactive oxygen spe-cies. Some NOX proteins have also been purified andstudied in (hyper)thermophilic organisms. NOX fromArchaeoglobus fulgidus may be involved in electrontransfer in sulfate respiration [7]. An H2O2-formingNOX functions as an alkyl hydroperoxide reductase inAmphibacillus xylanus [8]. Some NOX enzymes, suchas those of Pyrococcus furiosus [9] and Thermotogamaritima [10], have been proposed to protect anaer-obes from oxidative stress. In (hyper)thermophiles,the roles of some NOX enzymes remain to beelucidated [11].NADH oxidase varies with the organism; however,these proteins generally share similar secondary struc-tural folding [4,12]. An NOX from Thermus thermophi-lus is a homodimer as determined by X-raycrystallography [13]. Gel filtration chromatographyindicated that NADH:flavin oxidoreductase fromEubacterium is composed of three identical subunits[14]; NOX in Clostridium thermohydrosulfuricum isprobably made up of six subunits, as demonstrated bygel filtration [15]. In contrast, a heterogeneous NOXfrom Eubacterium ramulus is proposed to have an a8b4assembly, as revealed by gel filtration and PAGE [16].Two NOX enzymes from the Thermococcaceae fam-ily have been described. One is a novel enzyme inP. furiosus that produces both H2O2(77%) and H2O(23%) [9]. The other NOX, in Pyrococcus horikoshiiOT3, may function as a CoA disulfide reductase (CoA-DR) [17]; however, the function and structure of NOXin Thermococcus, a genus of the Thermococcaceae fam-ily, has not been clarified. In this study, we havecloned, overexpressed and purified a NOX that is com-posed of two cysteine moieties from T. profundus.Wereport its biochemical characterization and structure,and also used mutants to analyze its catalytic mecha-nism.ResultsCloning and sequencing of the nox gene fromT. profundusIn order to clone the T. profundus nox gene (NOXtp),we utilized a PCR-based DNA-walking method usingthe ClonTech genome-walker cloning kit, as describedin Experimental procedures; the resulting DNAsequence comprised an ORF of 1329 bp, predicting aprotein composed of 442 amino acids with a molecularmass of 48 611 Da. Figure 1 shows the nucleotidesequence of NOXtp and its flanking regions, togetherwith the translated amino acid sequence. The 5¢-flank-ing region of NOXtp contained a putative Archaeapromoter with a TATA box and ribosome-binding site.The 3¢-flanking region did not match with otherArchaea genes, as judged by homolog searches in theNCBI database. Unlike NOX, which has only oneconserved cysteine residue (Cys45) in its N-terminus[4], the amino acid composition of NOXtp revealedthe presence of two cysteine residues, Cys45 andCys122. Additionally, two conserved cofactor-bindingdomains were also identified in NOXtp. One was aFAD-binding domain containing the AMP-bindingand FMN-binding motifs observed in enzymes belong-ing to the glutathione reductase (GR) family [18]. Theother domain was a glycine-rich NAD-binding motiflocated between the AMP-binding and FMN-bindingmotifs (two FAD-binding domains) (Fig. 1). We pro-pose that NOXtp belongs to the GR family, becauseof the high sequence identity of the cofactor-bindingdomains described above.Multiple sequence alignment (Fig. S1) revealed thatCys45 is located at a similar position to that of thecysteine residue in the conserved active site of NOXfrom P. horikoshii (also called CoADR) [17] and NOXand NADH peroxidase from Enterococcus faecalis[19,20]. Sequence analysis by clustal w showed thatNOXtp shared a significant level of identity with NOX(CoADR) from P. horikoshii (80%) [17], NADH per-oxidase from E. faecalis (28%) [20], and NOX fromP. furiosus (36%) [9], Lactococcus lactis (30%) [21],Lactococcus sanfranciscensis (26%) [11] and E. faecalis(27%) [19] (Fig. S1). These proteins are generally com-posed of two identical subunits related by two-foldsymmetry. Each subunit can be divided into a C-termi-nal dimerization domain and an N-terminal pyridinenucleotide disulfide oxidoreductase domain, which isactually a small NADH-binding domain with a largeFAD-binding domain [4,12]. NOXtp has similarprimary structure architecture to these proteins asdetermined by NCBI protein blast analysis.Purification of native and recombinant NOXtpIn order to understand the oxygen detoxification mecha-nism of anaerobic microbes, we purified NOXtp fromT. profundus by several chromatographic methods. Thepurified protein revealed a subunit with a molecularmass of approximately 50 kDa (Fig. S2). The N-termi-nal amino acid sequence of purified NOXtp from T. pro-fundus was determined to be MERKRVVIIGGGAAG,Hexameric thermophilic NADH oxidase-producing H2O B. Jia et al.5356 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBSwhich is highly similar to that of NOX in T. kodakaren-sis KOD1, P. furiosus, Pyrococcus abyssi and P. horiko-shii OT3, belonging to the pyridine nucleotide disulfideoxidoreductase family. The purification of recombinantNOXtp from Escherichia coli was performed by ionexchange chromatography as described in Experimentalprocedures. SDS ⁄ PAGE analysis of recombinantNOXtp revealed a molecular mass of approximately50 kDa, which is close to that of the purified proteinfrom T. profundus (Fig. S2). However, gel filtrationanalysis under nondenaturing conditions showed thatthe purified NOXtp had a molecular mass ofapproximately 300 kDa (Fig. 2). These results indicatedthat NOXtp is a hexamer of 50 kDa subunits, incontrast to NOX proteins from thermophilic archaeans,which have been reported to be dimers or tetramers[13,17].Structure of NOXtpGel filtration analysis under nondenaturing conditionsrevealed that purified NOXtp has a molecular massof  300 kDa, corresponding to a hexamer with50 kDa subunits (Fig. 2). This structure is differentfrom that of other homologous NOX proteins, whichconsist of dimers or tetramers as revealed by X-raycrystallographic studies [12,13,22]. In order to clarifythe oligomeric structure of NOXtp, we performedelectron microscopy using purified NOXtp. The elec-tron micrographs of the negatively stained NOXtpoligomers showed a uniform distribution of the ring-shaped structure in the top-view orientation(Fig. 3A). In total, 939 well-stained particles weretranslationally aligned, and were subjected to multi-variate statistical analysis [23]. The eigenimagesFig. 1. Nucleotide sequence of the noxtpgene and predicted amino acid sequenceof the gene product from Thermo-coccus profundus. The putative TATA-boxand ribosome-binding site are underlinedand in bold letters, respectively. The resi-dues involved in FAD binding are shadowedin gray. The NAD-binding site is boxed. Thecysteine residues are in bold italic.B. Jia et al. Hexameric thermophilic NADH oxidase-producing H2OFEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5357obtained from the translationally, but not rotation-ally, aligned images revealed a six-fold rotationalsymmetry (Fig. 3Ba). Using the 10 most significanteigenvectors, nine classes were discriminated on thebasis of similarity of features after rotational align-ment without symmetrization. Most class averagesshowed a star-shaped structure with six-fold symme-try with heavy stain accumulation in its center(Fig. 3Bb). In particular, the two class averagesshown in panels 3 and 6 of Fig. 3Bb exhibited anobvious deviation from the star-like structure. Thiscould result from incomplete stain embedding of theparticle or from an unintentional inclination duringpreparation or microscopy. In order to analyzefurther the rotational symmetry of the top-on-viewimages, the same dataset was separated into manyclasses (10–30) using different eigenimages (10–20).The dataset was also aligned with an arbitrarilychosen reference and separated according to the simi-larity of features in the eigenimages. The resultingclass averages revealed no other statistically signifi-cant symmetry (data not shown). We found noFig. 2. Gel filtration chromatography profile of NOXtp purified fromEs. coli. The purified protein was subjected to Superdex-200 gel fil-tration chromatography. Absorbance was measured at 280 nm. Thex-axis shows the elution time. The standard proteins are ferritin(440 kDa), catalase (232 kDa), albumin (67 kDa) and ovalbumin(43 kDa).ABaCDbFig. 3. Electron micrograph and structuralanalysis of NOXtp. (A) Purified NOXtp wasabsorbed onto the grids as described inExperimental procedures. The electronmicrograph of the protein was then obtainedby negative staining with 2% uranyl acetate.(B) Multivariate statistical analysis of NOXtp.(a) The average (AV) of 939 translationally,but not rotationally, aligned particles withend-on orientation and the 10 most signifi-cant eigenimages (numbers 1–10) areshown. In (b), the nonsymmetrized classaverages (numbers 1–9) were derived fromrotationally aligned images using the 10most significant eigenvectors. The numeralsshown in the top right corner of the classaverages are the number of particles seenin each class. (C) The average of the side-onview of NOXtp (939 particles). (D) A sche-matic model for the assembly of NOXtpcomplexes. The diameters of the cavity,middle ring and outer ring are 4, 15 and19 nm, respectively.Hexameric thermophilic NADH oxidase-producing H2O B. Jia et al.5358 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBSevidence for the existence of a NOXtp protein withintrinsically lower symmetry, at least at the resolutionemployed.The average of the 939 top-on views revealed a star-shaped structure (Fig. 3C), which contained a middleregion of high density with heavy stain accumulationin its center. The average view also revealed that thedensity of the complex was not homogeneous; the den-sity increased towards the middle, such as seen in thevalosine-containing protein-like ATPase from Thermo-plasma acidophilum complex, which is composed oftwo stacked ring structures of different diameters [24].The upper (or middle) ring of the NOXtp complex hasa region that is denser than that of the outer ring, indi-cating the presence of a cavity in the complex with awidth of approximately 4 nm. The diameters of theouter ring and the middle ring (Fig. 3D) were approxi-mately 19 and 15 nm, respectively. These projectedimages as well as the gel filtration analysis indicatedthat NOXtp predominantly exhibits a hexameric star-shaped structure, in contrast to the structure recentlyreported by Kuzu et al. [22], which suggested a tetra-meric structure for NOX from Lactobacilluus brevis.NOXtp is an FAD-dependent NADH and NADPHoxidaseOn the basis of the amino acid sequence, NOXtp con-tains two FAD-binding domains. The isoalloxazinering system in FAD has been suggested to induce lightabsorbance in the UV and visible spectral range, givingrise to the yellow appearance of flavin and flavopro-teins [25]. We performed light absorbance analysis toconfirm NOXtp binding to FAD. Purified NOXtpfrom Es. coli has absorption maxima at 378 and456 nm, with a shoulder at 480 nm, which are charac-teristic spectral features of proteins with bound flavincofactors (Fig. 4A). The absorbance behavior alsoallowed the determination of the number of flavin mol-ecules bound per mole of NOXtp subunit [17,25,26]. Astoichiometry of 0.7–0.9 mol FAD per mol NOXtpsubunit was determined from the absorbance at460 nm.As NOXtp contains FAD as a prosthetic group,apo-NOXtp was prepared by hydrophobic interactionchromatography under acidic conditions (pH 3.5)with saturated NaBr buffer [26,27], in order to00.040.080.120.160.2300400 500 600 700 800 (nm)Absorbance01020304050607080901000 2 4 6 8 10 12 (min)NAD(P) amount (μM)01234567820 30 40 50 60 70 80 90 100 (ºC)Specific activity (U·mg–1)012345678Specific activity (U·mg–1)5678910 (pH)ABCDFig. 4. Activity assays of NADH and NADPH oxidase. (A) Visiblespectra of NOXtp (solid line), apo-NOXtp (dashed line) and theC45A mutant (dotted line). The absorbance was measured in50 mM sodium phosphate buffer (pH 7.2) at 25 °C. (B) FAD effecton NAD(P)H oxidase activity. An activity assay was performed asdescribed in Experimental procedures. The solid line shows theNADH oxidase activity of NOXtp purified from Es. coli (h), reconsti-tuted NOXtp (s), and apo-NOXtp (4). The dashed line shows theNADPH oxidase activity of NOXtp from Es. coli (h), reconstitutedNOXtp (s), and apo-NOXtp (4). (C) Optimal temperature of NAD(P)Hoxidase activity. The assay was performed at the indicated temper-atures in 50 mM potassium phosphate buffer (pH 7.2). NADH andNADPH oxidase activity are shown by a solid line and a dashedline, respectively. The squares show the measured temperaturepoints. (D) Optimal pH of NAD(P)H oxidase activity. Different bufferswere used in this assay. Sodium phosphate was used at pH 6.0,6.6, 7.2 and 7.7; Hepes buffer and Tris buffer were used at pH 8.0and 8.5; sodium borate buffer was used at pH 9.0. These bufferswere used at a concentration of 50 mM. NADH and NADPH oxi-dase activity are shown by a solid line and a dashed line, respec-tively. The squares show the measured pH points.B. Jia et al. Hexameric thermophilic NADH oxidase-producing H2OFEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5359confirm the function of FAD. The absorption spec-trum of apo-NOXtp did not show any significantabsorbance in the visible region, revealing that FADwas indeed absent (Fig. 4A). To determine whetherFAD was required for the enzymatic activity ofNOXtp, holoprotein and apoprotein activities wereassayed. The NADPH oxidase activity of NOXtpwas also measured, as described previously for NOX(CoADR) from P. horikoshii [17] and NOX fromL. sanfranciscensis [12], which show high similarity toNOXtp and accept both NADH and NADPH ascofactors. The activity of the reconstituted enzyme,which was accomplished by incubating equimolarconcentrations of apomonomers and FAD at roomtemperature for 5 min [26], was also measured. Theseassays revealed that NADH oxidase activity wasslightly higher than that of NADPH oxidase, andFAD significantly restored the oxidase activity ofapo-NOXtp (Fig. 4B). These results clearly indicatedthat NOXtp is an FAD-dependent NADH andNADPH oxidase, in contrast to NOX enzymes fromother thermophilic archaeons, which only exhibitactivity towards NADH [9–11].To further determine the function of NOXtp, thesteady-state kinetic parameters of NOXtp with eitherNADH or NADPH as the reducing substrate weremeasured at pH 7.2. NOXtp could catalyze NADHand NADPH oxidization with kcatvalues of6.2 ± 0.5 ⁄ s and 2.5 ± 0.3 ⁄ s, respectively. The steady-state kinetic parameters of NOXtp were similar tothose of NOX (CoADR) from P. horikoshii (Table 1).On the basis of the Km, both enzymes preferredNADPH as the substrate for oxidase activity, indicat-ing that NOX (CoADR) from P. horikoshii and NOX-tp belong to similar enzyme families. The optimaltemperature for the NADH and NADPH oxidaseactivity of NOXtp was near 70 °C (Fig. 4C), which islower than the optimal growth temperature (80 °C) ofthis organism. The optimal pH was between 7.5 and8.0 for both NADH and NADPH oxidase activity(Fig. 4D).NOXtp preferentially produces H2OThe product of O2reduction is an important factor inevaluating the physiological function of NOXs [10].For instance, NOX from P. furiosus, which may pro-tect anaerobic thermophiles against oxidative stress,can produce both H2O2and H2O [9]. In order todetermine the product of the NAD(P)H oxidase activ-ity of NOXtp, reactions containing 100 lm NAD(P)Hwere performed [all NAD(P)H consumed] according tothe published method [9], and H2O2was quantifiedusing a peroxi-DETECT kit from Sigma (St Louis,MO, USA). When NADPH oxidation was performedat 80 °C, approximately 7% of the NADPH suppliedwas used to produce H2O2, and 2% of the NADH wasrecovered as H2O2under the same conditions(Fig. 5D). These results demonstrated that NOXtpproduces predominantly H2O using NADH andNADPH as electron donors.Cys45 but not Cys122 functions as the nonflavinredox centerNADH oxidase in members of the Thermococcaceaeefamily, such as T. kodakaraensis KOD1, P. horrikoshii,P. abyssi and P. furiosus, have only one conserved cys-teine residue, Cys45; however, the sequence of NOXtp(Fig. 1) revealed that it contains two cysteine residues,Cys45 and Cys122. As cysteines are important residuesfor NOX enzyme activity, we replaced Cys45 andCys122 with alanines to analyze the function of thesetwo residues. After purification using the same methodas that used for the wild-type enzyme, the number ofcysteines in the three mutant enzymes (NOXtpC45A,NOXtpC122A and NOXtpC45A⁄ C122A) was exam-ined using Ellman’s method (Table 2). The singlemutants, NOXtpC45A and NOXtpC122A, containedabout one cysteine, and the double mutant containedno cysteines. These data confirmed the identity of themutants and also indicated that the nonmutated cyste-ine remained in its native state. The visible absorptionspectra showed that the three mutants containedtightly bound FAD (Fig. 4A, NOXtpC45A onlyshown – the other two mutants produced similarabsorbance spectra). Electron microscopy and nativePAGE showed no significant difference between wild-type NOXtp and its mutants (Fig. 5A,B). All of thedata indicated that the disulfide bond was not respon-sible for hexameric oligomerization and that substitu-tion of Cys45 and Cys122 with alanine did not resultin major changes in NOXtp quaternary structure.In order to determine the catalytic mechanism ofNOXtp, NAD(P)H oxidase assays were performedTable 1. Steady-state kinetic parameters of NOXtp and NOX (CoA-DR) from Pyrococcus horikoshii (50 mM potassium phosphate buf-fer, pH 7.2, 75 °C). Data shown are means of triplicatedeterminations ± SD.ParameterNOXtp-NADHoxidaseNOXtp-NADPHoxidaseCoADR-NADHoxidaseCoADR-NADPHoxidaseKm(lM) 53.1 ± 2.8 12.1 ± 1.1 73a13akcat(s)1) 6.2 ± 0.5 2.5 ± 0.3 8.2a2.0aaFrom reference [17].Hexameric thermophilic NADH oxidase-producing H2O B. Jia et al.5360 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBSwith the three mutants under the same conditions asused for the wild-type. The results showed that theC122A mutant had similar NADH and NADPH oxi-dase activity to that of the wild-type protein; however,the C45A mutant and the double C45A ⁄ C122Amutant had < 10% of the NAD(P)H oxidase activityof the wild-type protein (Fig. 5C). These results aresimilar to those obtained with a NOX from E. faecalis,where a serine substitution of its active site residueCys42 (C42S) resulted in approximately 3% of theactivity of the wild-type under the same conditions[4,28]. Considering these results, Cys45 may providethe essential second redox center in addition to the fla-vin. We further examined the products of NOXtp andits mutants. NAD(P)H oxidation was allowed to go tocompletion, and the amount of H2O2formed in thereaction was quantified using the peroxi-DETECT kit.The NOXtpC122A mutant produced a similar amountof H2O as the wild-type under the same conditionsand with the same substrates (Fig. 5D). Oxidation ofNADH and NADPH by NOXtpC45A and NOX-tpC45A ⁄ C122A led to the formation of about oneequivalent of H2O2(Fig. 5D), demonstrating thatH2O2production by these two mutants is stoichiome-tric with NADH and NADPH oxidation. The activityand product assays using the wild-type and mutantsclearly demonstrated that Cys45 participates in thedirect four-electron reduction of O2to H2O, and theCys45 mutation alters the reaction to produce H2O2instead of H2O.DiscussionIn this study, we have demonstrated that NOXtp has ahexameric ring-shaped structure. Gel filtration undernondenaturing conditions revealed that NOXtp is com-posed of six subunits. Moreover, upon electron micro-scopic analysis, NOXtp was found to predominantlyexhibit a hexameric structure that contained a middleregion of high density with heavy stain accumulationin its center. However, the crystal structure of NOXfrom L. sanfranciscensis revealed a dimeric form withan N-terminal oxidoreductase domain and a C-termi-nal dimerization domain [12]. NPX from Streptococ-cus faecalis, catalyzing the conversion of H2O2toH2O, was reported to be a homotetrameric structure[29]. These two mesophilic proteins show differenttypes of subunit oligomerization and low sequenceidentity (Fig. S1), but each of their subunits showshigh structural similarity and their folding patterns aresimilar to that of GR [12,29]. In contrast, NOX fromThermoanaerobium brockii was found to have a hexa-meric quaternary structure by gel filtration [15]. Elec-tron microscopic analysis has revealed that NOXtp hasa hexameric ring-shaped structure composed of two012345678Specific activity (U·mg–1)12dcbadcba100 nm 100 nm100 nm100 nmabcd020406080100H2O2 production (µmol)12dcbadcba123 45669 kDa44067140232ABCDFig. 5. Comparisons of activity, products and structures between NOXtp and the mutants. (A) Electron micrographs of NOXtp (a), NOX-tpC45A (b), NOXtpC122A (c) and NOXtpC45A ⁄ C122A (d). The bar represents 100 nm. (B) Native PAGE of wild-type NOXtp and its mutants.Lanes 1–4 correspond to (a), (b), (c) and (d) in (A), respectively; lane 5 is the molecular weight marker. The lower part shows the correspond-ing proteins determined by SDS ⁄ PAGE. (C) Specific activity of wild-type NOXtp (a), NOXtpC45A (b), NOXtpC122A (c) and NOX-tpC45A ⁄ C122A (d) with NADH (bar 1) and NADPH (bar 2) as substrates. (D) The amount of H2O2produced by NOXtp (a), NOXtpC45A (b),NOXtpC122A (c) and NOXtpC45A ⁄ C122A (d) when 100 lM NADH (bar 1) or 100 lM NADPH (bar 2) was oxidized.Table 2. Determination of the sulfhydryl contents of wild-type andmutant NOXtp using Ellman’s reagent. Data shown are means oftriplicate determinations ± SD.ProteinNo. cysteinesper proteinNOXtp 1.84 ± 0.31NOXtpC45A 0.85 ± 0.34NOXtpC122A 0.84 ± 0.18NOXtpC45A ⁄ C122A 0.14 ± 0.07B. Jia et al. Hexameric thermophilic NADH oxidase-producing H2OFEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5361stacked rings of different diameters (19 and 15 nmrespectively) that encompass a central opening; this isthe first hexameric NOX determined by electronmicroscopy. Significantly, this structural feature ofNOXtp is highly similar to that of valosine-containingprotein-like ATPase from Th. acidophilum, an archaealmember of the AAA family (ATPases associated witha variety of cellular activities) [24]. In addition, thestructure of the cysteine mutants, NOXtpC45A, NOX-tpC122A and NOXtpC45A ⁄ C122A, was the same asthat of the wild-type. Thus, it appears that a disulfidebond does not participate in the oligomerization andquaternary structure of NOXtp.NADH oxidase catalyzes the transfer of electronsfrom reduced pyridine nucleotides to O2[2,4]. Here wehave demonstrated that NOXtp can efficiently reduceO2to produce H2O using both NADH and NADPHas electron donors. In addition, the activity and prod-uct assays of the wild-type and mutants showed thatCys45 is the active site residue and that Cys122 doesnot function in the NADH and NADPH oxidaseactivity. These results indicate that Cys45 participatesin the direct four-electron transfer reduction of O2toH2O, and that the Cys45 mutant alters the reductionto produce H2O2instead of H2O, similar to NOX inE. faecalis [28]. NOX in E. faecalis belongs to a groupof enzymes that use a cysteine sulfenic acid as the non-flavin redox center. These enzymes are found inEnterococcus and Streptococcus, which are aerotolerantanaerobes, where they play an important role in O2tolerance [4]. For example, H2O-forming NOX-defi-cient mutants of Streptococcus pyogenes are unable togrow under high-O2conditions, revealing the impor-tance of NOX-scavenging activity against harmful O2[30]. We therefore propose that NOXtp may decom-pose O2in the anaerobe T. profundus.The predominant production of H2O by NOXtp isin contrast to the exclusive production of H2O2bymost NOXtp homologs in thermophiles, such as NOXin A. fulgidus, Desulfovibrio gigas, Thermot. maritimaand Thermoanaerobium brockii [10,11,15,31]. Previously,the production of H2O2was considered to be the dis-tinctive property of NOX proteins from thermophiles[10,11], with the exception of NOX from P. furiosus,which produces both H2O2(77%) and H2O (23%) [9].To our knowledge, NOXtp is the first NOX to bepurified from thermophilic microorganisms that cancatalyze electron transfer from NADH and NADPHto O2and predominantly produce H2O. NOXtp istherefore better for removing O2than other reportedO2-scavenging systems, which must employ intermedi-ates to reduce H2O2produced by NAD(P)H oxidases,such as in D. gigas, where rubredoxin and neelaredoxinact as intermediates [31]. As NOXtp and the mesophil-ic enzymes that decompose injurious O2belong to thesame group (discussed above), and NOXtp reduces O2to H2O directly, we propose that NOXtp may play animportant role in O2removal or aerobic tolerance inthermophilic anaerobes.Experimental proceduresPurification of NOXtp from T. profundusThermococcus profundus cells (8 L) were grown at 80 °Casreported previously [32]. After harvesting, the cells were dis-solved in 20 mm potassium phosphate buffer (pH 6.5), con-taining 5 mm MgCl2, 0.5 mm EDTA, 1 mm dithiothreitoland 10% glycerol (PMEDG buffer), and disrupted by soni-cation. The homogenates were centrifuged at 10 000 g for30 min. The supernatant was loaded on a phosphocellulosecolumn that had been equilibrated with PMEDG buffer.After being washed completely, the proteins were eluted by100, 200, 300, 400, 500 and 1000 mm NaCl in a stepwisegradient, and the eluates in 200 mm NaCl were dialyzedwith 50 mm Tris buffer (pH 8.0) containing 400 mm NaCl.The sample was then loaded on an amino-benzimide col-umn equilibrated with the same buffer. Unabsorbed pro-teins on the resin were collected and dialyzed with PMEDGbuffer, concentrated using a centricon (Millipore, Billerica,MA, USA), and stored at )80 °C. Eluates in all steps werechecked by transmission electron microscopy. The proteinconcentration was determined by the Bradford method, andBSA was used as standard.SDS/PAGE and N-terminal sequencingThe purified enzyme was subjected to SDS ⁄ PAGE and elec-troblotted onto poly(vinylidene difluoride) membranes. Thevisible band was excised and applied to a protein sequenceanalyzer (Korea Basic Science Institute, Daejeon, Korea).Cloning of NOXtp from T. profundusPolymerase chain reaction experiments with T. profundusgenomic DNA as a template were performed using degener-ate oligonucleotides (sense primer, 5¢-GTA GTA ATAATA GGA GGA GGA GCN GCN GGN ATG-3¢; anti-sense primer, 5¢-TAN ACT TTY TCN CAN SWN GTYTGC AT-3¢; N = A, G, C and T; Y = C and T; W = Aand T). The sense primer was designed from the knownN-terminal sequence, and the antisense primer was fromthe conserved C-terminal sequence of NOX. The experi-ment using the two oligonucleotides afforded an amplificateof  1.3 kb, which was ligated into the pTOPO vector(Invitrogen, Carlsbad, CA, USA), transformed, andconfirmed by sequencing. The resulting sequence was usedHexameric thermophilic NADH oxidase-producing H2O B. Jia et al.5362 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBSfor subsequent cloning. Full gene cloning of NOXtp was per-formed using the Universal Genomewalker kit (ClonTech,Mountain View, CA, USA). Briefly, the genomic DNA wasdigested with EcoRV, DrabI, PvuII and SspI separately, andligated to the adaptor provided by the kit. PCR was per-formed with the adaptor primers (provided by the kit) andtail-specific primers (N-terminal genomewalker primer,5¢-TGG AGG TCT TTG CCG CGC TTT TTG AT-3¢;C-terminal genomewalker primer, 5¢-GGT GTG CAGGCT GTA AAT GCC GAG AT-3¢), which correspondedto the known sequence detected by the degenerate primers.The PCR products were ligated into the pTOPO vector,transformed, and sequenced.Expression and purification of NOXtp in Es. coliThe new primers (sense primer, 5¢-CGCGCG CCATGG AGAGGAAACGCGTTGTTAT-3¢; antisense primer,5¢-CGCGAAGCTT TAAAACTTTAGAACCCTG-3¢)weredesigned on the basis of the sequence of the Genomewalkerresult (the underlined bases indicate the restriction enzymesite). The PCR product and pET28-(a) were digested byNcoI and HindIII and ligated. The ligation product wastransformed into Es. coli BL21(DE3) by electroporation.Finally, the recombinant vector (pENOXtp) was confirmedby sequencing.Recombinant Es. coli cells (2 L) were cultured in LBbroth to a D600 nmof 1.0, and then induced with 1 mm iso-propyl-thio-b-d-galactoside for 4 h. After harvesting, thecells were resuspended in PMEDG buffer and disrupted bysonication. After centrifugation (3000 g, 30 min), the super-natants were heated at 65 °C for 30 min, and then thedenatured proteins were removed by centrifugation (3000 g,30 min). The supernatants were loaded onto a phosphocel-lulose column that had been equilibrated with the samebuffer. After being washed completely, the proteins wereeluted with 200 mm NaCl. The purified protein waschecked by SDS ⁄ PAGE and dialyzed with PMEDG buffer,concentrated, and stored at )80 °C.MutagenesisThe primers used for the single cysteine to alanine mutantswere as follows: C45A, forward primer, 5¢-ACG GAATGG GTG AGC CAC GCT CCC GCC GGT ATC CCCTAC GTA GTT GAG GGT-3¢; C45A, reverse primer,5¢-ACC CTC A AC TAC GTA GGG G AT ACC GGC GGGAGC GTG GCT CAC CCA TTC CGT-3¢; C122A, for-ward primer, 5¢-CCG CAG GTT CCG GCG ATA GAGGGC GCC CAC CTG GAA GGA GTA TTC ACA GCA-3¢; and C122A reverse primer, 5¢-TGC TGT GAA TACTCC TTC CAG GTG GGC GCC CTC TAT CGC CGG AACCTG CGG-3¢. The PCR was performed using Pfu polymer-ase (Takara, Kyoto, Japan), and the cycling parameterswere: 95 °C for 5 min (one cycle), 95 °C for 30 s, and 68 ° Cfor 12 min (12 cycles). After amplification, the PCR mixturewas digested with DpnI and then transformed into Es. coliBL21(DE3) by electroporation. The mutants were confirmedby DNA sequencing. The double cysteine mutants wereproduced by the same method, except that pENOXtpC45Awas used as the template and C122A primers were used forthe amplification. The mutant proteins were purified usingthe same method as used for wild-type purification.Gel filtration chromatographyThe sample (1 mgÆmL)1) was loaded onto a Superdex-200column (Amersham Biotech, Piscataway, NJ, USA). Stan-dard proteins included ferritin (440 kDa), catalase(232 kDa), albumin (67 kDa) and ovalbumin (43 kDa).Apo-NOX preparationThe purified NOXtp from Es. coli is a holoenzyme withFAD. The protein was dialyzed with 100 mm phosphatebuffer (pH 7.2) containing 2.4 m (NH4)2SO4,1mm dith-iothreitol and 0.5 mm EDTA, and then loaded on thehydrophobic interaction chromatography column equili-brated with the same buffer. FAD was eluted with equili-bration buffer saturated with NaBr (pH 3.5). The columnwas balanced again with the equilibration buffer, and theapoprotein was eluted with 100 mm phosphate buffer[26,27]. Eluates were dialyzed with the PMEDG bufferdescribed above, and stored at )80 °C.Enzyme assaysThe NADH or NADPH oxidase activity of the recombi-nant protein was examined by time-dependent removal ofNAD(P)H in aerobic conditions. The assays were per-formed in 50 mm sodium or potassium phosphate buffer(pH 7.2), 0.5 mm NAD(P)H and 100 mm NaCl at the indi-cated temperatures. The reaction was started by addingNOXtp in the amounts indicated. The rate of NAD(P)Hconsumption was measured by monitoring the decrease inA340 nm. One unit of activity was defined as the amount ofenzyme catalyzing the oxidation of 1 lmol NADH permin at 75 °Cin50mm potassium phosphate buffer(pH 7.2) and 0.5 mm NADH. To measure kinetic parame-ters, reaction rates were measured at a series of NAD(P)Hconcentrations, and the rates at various substrate concen-trations were finally fitted by Lineweaver–Burk plots. Theparameters (with standard deviation) were determined bythree separate experiments.Determination of the sulfhydryl contentThe sulfhydryl contents were determined using Ellman’sreagent in anaerobic conditions according to a publishedB. Jia et al. Hexameric thermophilic NADH oxidase-producing H2OFEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5363method [17,33]. After the proteins and 5,5¢-dithiobis-(2-nitrobenzoic acid) (Nbs2) were incubated for 15 min at25 °C, A412nm was monitored to estimate the number ofcysteine residues present as protein ⁄ Nbs2mixed disulfide.The sulfhydryl concentrations in these proteins were deter-mined from a calibration curve created using known con-centrations of standard l-cysteine solutions.H2O2detectionH2O2was detected using the PeroxiDetect Kit (Sigma).Briefly, the assay was performed in 50 mm sodium phos-phate buffer (pH 7.2), 100 lmol NAD(P)H, 1 mm EDTA,100 mm NaCl and 0.2 nmol NOXtp. The reaction wasallowed to go to completion. Reaction buffer (100 lL) wasadded to the kit. Peroxides convert Fe2+to Fe3+ionsunder acidic conditions. Fe3+ions will then form a coloredadduct with xylenol orange, which is observed at 560 nm.NAD(P)H will interfere with the H2O2assay, so all of theNAD(P)H must be consumed completely.Electron microscopy and image processingPurified NOX was applied to glow-discharged carbon-coated copper grids. After the proteins had been allowed toabsorb for 1–2 min, the grids were rinsed with droplets ofde-ionized water, and stained with 2% (w ⁄ v) uranyl acetate.Electron micrographs were recorded with an FEI TECH-NAI 12 microscope at a magnification of ·51 600 (nominalmagnification of ·52 000) and an acceleration voltage of120 kV.Light-optical diffractograms were used to select themicrographs, to examine the defocus and to verify that nodrift or astigmatism was present. Suitable areas were digi-tized as arrays of 1024 · 1024 pixels with leaf scan 45 ata pixel size of 20 lm, corresponding to 0.38 nm at the spec-imen level. For image processing, the semper [34] and em[35] software packages were used. From digitized micro-graphs, smaller frames of 64 · 64 pixels containing individ-ual particles were extracted interactively. These images werealigned translationally and rotationally, using standard cor-relation methods [36,37]. An arbitrarily chosen referencewas used for the first cycle of alignment and averaging, andthe resulting average was used as a reference in the secondrefinement cycle. For analysis of the rotational symmetry oftop-on-view images, the individual images were alignedtranslationally but not rotationally [38]. These alignedimages were subjected to multivariate statistical analysis[39]. The resulting eigenimages represent all-importantstructural features of the original dataset. If the images haddifferent rotational symmetries in the original dataset, theeigenimages would reveal the different symmetry axes.Moreover, these images can be distinguished and subse-quently separated on the basis on eigenimages. The rota-tionally aligned images were classified on the basis ofeigenvector–eigenvalue data analysis, and subsequent aver-aging was performed for each class separately. The averagewas finally symmetrized on the basis of angular correlationcoefficients [40].AcknowledgementsB. Jia, S. Lee, B. P. Pham, R. Yu and T. L. Le weresupported by scholarships from the Brain Korea21project in 2008, Korea. This work was supported by agrant from the MOST ⁄ KOSEF to the EnvironmentalBiotechnology National Core Research Center (grantno. R15-2003-012-01003-0), and the Korea ResearchFoundation Grant funded by the Korean Government(MOEHRD) (grant no. KRF-2007-521-C00241), toG. W. 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W, Dahlmann B, Hegerl R, Kopp F, Luehn L & Pfeifer G (1988) Electron microscopy and image analysis of the multicatalytic proteinase FEBS Lett 241, 239–245 37 Kim KI, Cheong GW, Park SC, Ha JS, Woo KM, Choi SJ & Chung CH (2000) Heptameric ring structure of the heat-shock protein ClpB, a protein-activated ATPase in Escherichia coli J Mol Biol 303, 655–666 38 Marco S, Urena D, Carrascosa JL, Waldmann . was also measured. Theseassays revealed that NADH oxidase activity wasslightly higher than that of NADPH oxidase, andFAD significantly restored the oxidase. Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H2OBaolei Jia1,*, Seong-Cheol Park1,*,, Sangmin
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Xem thêm: Báo cáo khoa học: Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H2O pot, Báo cáo khoa học: Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H2O pot, Báo cáo khoa học: Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H2O pot