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• W2044903071 abstract "A NAD(P)H oxidase has been isolated from the archaeon Sulfolobus solfataricus. The enzyme is a homodimer with M r 38,000 per subunit (SsNOX38) containing 1 FAD molecule/subunit. It oxidizes NADH and, less efficiently, NADPH with the formation of hydrogen peroxide. The enzyme was resistant against chemical and physical denaturating agents. The temperature for its half-denaturation was 93 and 75 °C in the absence or presence, respectively, of 8m urea. The enzyme did not show any reductase activity. TheSsNOX38 encoding gene was cloned and sequenced. It accounted for a product of 36.5 kDa. The translated amino acid sequence was made of 332 residues containing two putative βαβ-fold regions, typical of NAD- and FAD-binding proteins. The primary structure of SsNOX38 did not show any homology with the N-terminal amino acid sequence of a NADH oxidase previously isolated from S. solfataricus (SsNOX35) (Masullo, M., Raimo, G., Dello Russo, A., Bocchini, V. and Bannister, J. V. (1996) Biotechnol. Appl. Biochem. 23, 47–54). Conversely, it showed 40% sequence identity with a putative thioredoxin reductase from Bacillus subtilis, but it did not contain cysteines, which are essential for the activity of the reductase. A NAD(P)H oxidase has been isolated from the archaeon Sulfolobus solfataricus. The enzyme is a homodimer with M r 38,000 per subunit (SsNOX38) containing 1 FAD molecule/subunit. It oxidizes NADH and, less efficiently, NADPH with the formation of hydrogen peroxide. The enzyme was resistant against chemical and physical denaturating agents. The temperature for its half-denaturation was 93 and 75 °C in the absence or presence, respectively, of 8m urea. The enzyme did not show any reductase activity. TheSsNOX38 encoding gene was cloned and sequenced. It accounted for a product of 36.5 kDa. The translated amino acid sequence was made of 332 residues containing two putative βαβ-fold regions, typical of NAD- and FAD-binding proteins. The primary structure of SsNOX38 did not show any homology with the N-terminal amino acid sequence of a NADH oxidase previously isolated from S. solfataricus (SsNOX35) (Masullo, M., Raimo, G., Dello Russo, A., Bocchini, V. and Bannister, J. V. (1996) Biotechnol. Appl. Biochem. 23, 47–54). Conversely, it showed 40% sequence identity with a putative thioredoxin reductase from Bacillus subtilis, but it did not contain cysteines, which are essential for the activity of the reductase. NADH oxidase Sulfolobus solfataricus, Sa, Sulfolobus acidocaldarius Thermus aquaticus Thermus thermophilus 5,5′-dithiobis-(2,2′-dinitrobenzoate) polyacrylamide gel electrophoresis 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid high pressure liquid chromatography polymerase chain reaction In several bacterial cells, the NADH formed under aerobic conditions by various dehydrogenases is converted to NAD+by NADH oxidase (NOX),1 which is considered responsible for the maintenance of the intracellular redox balance (1.Niimura Y. Ohnishi K. Yarita Y. Hidaka M. Masaki H. Uchimura T. Suzuki H. Kozaki M. Uozumi T. J. Bacteriol. 1993; 175: 7945-7950Crossref PubMed Google Scholar, 2.Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar). The enzyme has been isolated and characterized from various mesophilic and thermophilic eubacteria (3.Masullo M. Ph.D. thesis. Cranfield University, Cranfield, UK1996Google Scholar). There are two types of NADH oxidase; one catalyzes the four-electron reduction of O2 with formation of H2O, and the other catalyzes the two-electron reduction of O2 to H2O2. The latter is found in many microorganisms including thermophilic eubacteria. We (4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar) have previously reported the isolation and characterization of NADH oxidase from the archaea Sulfolobus solfataricus andSulfolobus acidocaldarius. The S. solfataricusenzyme is a homodimer composed of two subunits ofM r 35,000 (SsNOX35) and contains 1 mol of FAD/subunit, whereas the S. acidocaldarius enzyme is a monomer of M r 27,000 (SaNOX27), which is purified without bound flavin nucleotide. Both enzymes are H2O2-forming NADH oxidases. Furthermore, the N-terminal amino acid sequences of the two enzymes do not show any sequence homology, neither between themselves nor with the amino acid sequence of other NADH oxidases (3.Masullo M. Ph.D. thesis. Cranfield University, Cranfield, UK1996Google Scholar). In contrast, the N-terminal sequence of the first 23 amino acid residues of SaNOX27 showed 57% identity with a β-alanine-piruvate aminotransferase (5.Matsui-Lee I.S. Muragaki Y. Ideguchi T. Hase T. Tsuji M. Ooshima A. Okuno E. Kido R. J. Biochem. (Tokyo). 1995; 117: 856-862Crossref PubMed Scopus (23) Google Scholar) and 47% identity with a putative NADP reductase from the archaeonMethanococcus jannaschii (6.Bult C.J. White O. Olsen Zhou L. Fleischmann R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Dougherty B.A. Tomb J.F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S.M. Venter J.C. GGJScience. 1996; 273: 1058-1073Crossref PubMed Scopus (2284) Google Scholar). In this work, we report the purification, the biochemical characterization, and the cloning of the gene of a second H2O2-forming NAD(P)H oxidase from the archaeonS. solfataricus (SsNOX38). The relationship of the primary structure of this enzyme with that of other NADH oxidases and oxidoreductases is also analyzed. Restriction enzymes, modifying enzymes, and labeled compounds were from Amersham Pharmacia Biotech. Plasmid DNA, genomic DNA, and labeled probes were prepared as described (7.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning : A Laboratory Manual,2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). β-NADH, β-NADPH, α-NADH, deamino NADH, FAD, ATP, 4-aminoantipyrine, 2,6-dichloroindophenol, DTNB, and peroxidase were from Sigma. The following buffers were used: buffer A (25 mm MES/KOH, pH 5.5); buffer B (20 mm Tris/HCl, pH 7.8; 5 mmMgCl2, 50 mm KCl, 200 μmphenylmethylsulfonyl fluoride); buffer C (20 mm Tris/HCl, pH 7.8); and buffer D (20 mm MOPS, pH 7.0, 5 mmCH3COONa, 1 mm EDTA). NADH oxidase activity was evaluated spectrophotometrically at 65 °C by determining the initial rate of NADH oxidation in capped cuvettes. The reaction mixture contained 0.18 mm FAD, 1 mm NADH (or NADPH) in 1 ml final volume of buffer A. The reaction was started by adding the enzyme, and the reaction was followed kinetically by measuring the decrease of absorbance at 340 nm (ε = 6220m−1·cm−1). One unit of enzyme was defined as the amount of enzyme that catalyzes the oxidation of 1 μmol of substrate/min at 65 °C. Blanks without the addition of the enzymes were run to evaluate the spontaneous oxidation of substrate at 65 °C. The determination of the hydrogen peroxide produced by the NADH oxidase was performed as described (8.Thurman R.G. Ley H.G. Scholtz R. Eur. J. Biochem. 1972; 25: 420-430Crossref PubMed Scopus (479) Google Scholar). The reaction was carried out in 0.25 mm phosphate buffer, pH 7.2, 0.25 mm 4-amino antipyrine, 25 mm phenol and 0.2 units of peroxidase. After a 30-min incubation at room temperature, the absorbance was measured at 500 nm, and the concentration of H2O2 was calculated from a calibration curve. Assays for the DTNB and 2,6-dichloroindophenol reductase activities of NADH oxidase were performed as already described (2.Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar). The reaction mixture contained 0.3 mm EDTA, 75 μm2,6-dichloroindophenol or 0.4 mm DTNB, and 1 mmNADH in 1 ml final volume of 50 mm phosphate buffer, pH 7.0, The reaction was started by adding 3 μg of purifiedSsNOX38. The absorbance was monitored at 600 nm for the 2,6-dichloroindophenol and at 412 nm for DTNB. S. solfataricus cells (strain MT-4, ATCC 49255) were grown as already reported (9.De Rosa M. Gambacorta A. Nicolaus B. Giardina E. Poerio E. Buonocore V. Biochem. J. 1984; 224: 407-414Crossref PubMed Scopus (141) Google Scholar). 80 g of cells were resuspended in 300 ml of buffer B. The mixture was frozen and thawed twice, and ground with 100 g of sand for 15 min at 4 °C. From this point, all of the purification steps were carried out at 4 °C. The suspension was centrifuged at low speed to remove the sand, sonicated for 5 min on ice at 100 W, and centrifuged at 100,000 × g to remove cell debris. The S-100 fraction (262 ml) was then dialysed against buffer C containing 10% glycerol and was applied on a DEAE-Sepharose fast-flow (2.5 × 80 cm) equilibrated in the same buffer at 2 ml/min. The column was then washed with buffer C containing 10% glycerol, and 22-ml fractions were collected. NADH oxidase activity was assayed on 450-μl aliquots, and the activity was eluted in two peaks corresponding to fractions 15–40 (pool I) and 41–80 (pool II), respectively. Pool II was concentrated by Aquacide II to 60 ml, dialysed against buffer C, loaded onto a Affi-gel blue column (1 × 8 cm, flow-rate 1 ml/min), washed with buffer C, and eluted with a 400-ml linear gradient 0–300 mm KCl in buffer C; 10 ml fractions were then collected. The enzyme activity, assayed on 50-μl aliquots, was eluted at 150–210 mm KCl. The pool of active fractions was concentrated by Aquacide II to about 3 ml and applied to a Superdex-75 gel filtration column (2.6 × 60 cm, flow-rate 1 ml/min), and eluted with buffer C containing 100 mm NaCl, and 4-ml fractions were collected. NADH oxidase activity was assayed on 25-μl aliquots. Active fractions, showing a single band on SDS-PAGE, were pooled, concentrated by Aquacide II, dialysed against buffer C containing 100 mm NaCl, 50% glycerol, and stored at −20 °C. Trypsin digestion of SsNOX38 was performed in buffer B at an enzyme concentration of 0.3 mg/ml in the presence of 0.1 mg/ml trypsin. The mixture was incubated at 37 °C, and at different times aliquots were withdrawn, and the reaction was stopped by adding soybean trypsin inhibitor (25 μg/ml final concentration) and finally analyzed on 14% SDS-PAGE. K m andV were determined by measuring the initial rate of NADH consumption at different substrate concentrations and in the presence of 0.2 mm FAD. The affinity of the enzyme for the cofactor was determined at different concentrations of FAD in the presence of saturating NADH concentration. Values of K m andV were calculated by Lineweaver-Burk plots. The heat inactivation of SsNOX38 was followed kinetically by incubating 1 mg/ml protein in buffer C at selected temperatures. After the heat treatment, aliquots were withdrawn, cooled on ice for 30 min, and then analyzed for the residual NADH oxidase activity as described above. UV melting curves of SsNOX38 were obtained in the temperature range of 50–105 °C using a computer-assisted Cary 1E spectrophotometer (Varian) equipped with a temperature controller. The increase in temperature was set at 0.1 °C/min, and the difference in absorbance at 286 and 274 nm was measured every second degree centigrade increase, normalized between 0 and 100, and plottedversus temperature (10.Arcari P. Masullo M. Arcucci A. Ianniciello G. de Paola B. Bocchini V. Biochemistry. 1999; 38: 12288-12295Crossref PubMed Scopus (18) Google Scholar). The thermophilicity ofSsNOX38 was evaluated in the temperature range of 50–100 °C. At each temperature the reaction was followed spectrophotometrically as described above. A molecular probe for the isolation of the SsNOX38 gene was synthesized by PCR using as primer two oligonucleotides derived from the amino acid sequence of SsNOX38 and as template the S. solfataricus DNA. The forward primer, ATG·GAT·GGA·TAT·GAT·ATA·GT (nox1), was deduced from the N-terminal sequence MDEYDIV of the purified protein, whereas the reverse primer, TC·ATG·CCA·TAC·ATA·TAC·ATT (nox2), was designed from the N-terminal sequence NVYVWH of a 14-kDa tryptic fragment of the enzyme. Degenerations on the third codon position were reduced to a single nucleotide (indicated in bold) on the basis of the preferential codon usage in S. solfataricus(11.De Vendittis E. Bocchini V. Gene. 1996; 176: 27-33Crossref PubMed Scopus (17) Google Scholar). A S. solfataricus DNA library was prepared into the pUC18 cloning vector, upon digestion of both DNAs withEcoRI/PstI restriction enzymes. About 2 × 103 colonies were analyzed using as probe the32P-labeled PCR fragment. Electrophoretic analysis was performed on 14% polyacrylamide gels in the presence of SDS (12.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar) using appropriateM r standards. Protein concentration was determined according to Bradford (13.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215653) Google Scholar). Nucleotide sequencing of theSsNOX38 gene was performed on both DNA strands using the T7 sequencing kit (Promega) and synthetic oligonucleotides as primers. Southern blot on S. solfataricus genomic DNA was performed as described (14.Southern E. J. Mol. Biol. 1975; 98: 503-517Crossref PubMed Scopus (21463) Google Scholar). The amino acid sequence of the N-terminal region and that of a tryptic fragment of the protein were determined by automated Edman degradation on a pulsed liquid sequencer (Applied Biosystems) connected on-line to a HPLC apparatus for PTH-amino acid identification. A query for sequence similarities was addressed to the GenBankTM data base using the BLASTP program (15.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Mol. Biol. 1990; 215: 403-410Crossref Scopus (70353) Google Scholar). Sequence analyses and alignments were established with the help of a nucleic acid and protein analysis software system packed with the CLUSTAL program (16.Higgins D.G. Sharp P.M. Comput. Appl. Biosci. 1989; 5: 151-153PubMed Google Scholar). The SsNOX38 purification procedure is summarized in Table I. After the DEAE-Sepharose fast-flow step, two peaks of NADH oxidase activity were found in the flow-through (not shown). The first peak (pool I) led to the purification of a 35-kDa NADH oxidase (SsNOX35) as described in a previous work (4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar), whereas the second peak (pool II) led to the purification of the SsNOX38. The enzyme activity was purified 187-fold with a recovery of 21%. The final product was homogeneous on both SDS-PAGE and HPLC C4 column.Table IPurification of SsNOX38 from 80 g of S. solfataricus cellsTotal proteinTotal activitySpecific activityYieldPurificationmgunitunit/mg%-foldS-1003511540.0151001DEAE53.1320.65940Affi-gel blue11.418.31.634107Superdex 754.011.22.821187One unit is defined as the amount of enzyme catalysing the oxidation of 1 μmol of NADH/min at 65 °C. Open table in a new tab One unit is defined as the amount of enzyme catalysing the oxidation of 1 μmol of NADH/min at 65 °C. The molecular mass ofSsNOX38 as estimated by SDS-PAGE was about 38 kDa (Fig.1), whereas the molecular mass determined by gel filtration on a Superdex-75 HR 10/30 was about 70 kDa (not shown). These data suggested that the native enzyme is made of two identical subunits not covalently linked. The absorption spectrum of the enzyme was typical of a flavoprotein, with a major peak at 275 nm and two minor peaks at 375 and 470 nm (Fig.2 A). The fluorescence spectrum showed a maximum at about 520 nm (Fig. 2 B). These features suggest that, like SsNOX35 (4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar), even purifiedSsNOX38 is bound to a flavin cofactor.Figure 2Spectroscopic properties ofSsNOX38. Absorption (A) and fluorescence (B) spectra recorded at 25 °C at a protein concentration of 0.1 mg/ml in 20 mm Tris·HCl buffer, pH 7.8. In B, the excitation wavelength was 490 nm, and both the excitation and emission slits were set at 10 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) SsNOX38 catalyzed the oxidation of NADH in the presence of molecular oxygen with formation of hydrogen peroxide. A stoichiometric amount of H2O2 was produced upon complete oxidation of NADH (Fig. 3), thus suggesting that the enzyme transfers two electrons from NADH to molecular oxygen. The enzyme required the presence of FAD as cofactor. NADPH was also a substrate for the enzyme, but it was oxidized at a rate about twice as slow as NADH. Furthermore, the enzyme did not exhibit activity toward α-NADH and deamino NADH (Fig.4). The kinetic parameters of the reaction using NADH as substrate and FAD as cofactor are summarized in Table II and compared with those of other thermophilic NADH oxidases (2.Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar, 17.Park H.J. Reiser C.O.A. Kondruweit S. Erdmann H. Schmid R.D. Sprinzl M. Eur. J. Biochem. 1992; 205: 881-885Crossref PubMed Scopus (111) Google Scholar, 18.Cocco D. Rinaldi A. Savini I. Cooper J.M. Bannister J.V. Eur. J. Biochem. 1988; 174: 267-271Crossref PubMed Scopus (45) Google Scholar, 19.Maeda K. Truscott K. Liu X.L. Scopes R.K. Biochem. J. 1992; 284: 187-190Crossref Scopus (29) Google Scholar). The enzyme was not able to catalyze the electron transfer from NADH to 2,6-dichloroindophenol or to DTNB, typical electron acceptors of NADH dehydrogenases and thiol disulfide oxidoreductases activity, respectively.Figure 4Substrate specificity ofSsNOX38. The oxidase reaction was carried out at 65 °C in buffer A in the presence of 0.2 mm FAD with the indicated amounts of purified SsNOX38 and 1 mmNADH (●), NADPH (▪), deamino NADH (▴), or α-NADH (♦). Blanks caused by the spontaneous oxidation of the substrates were subtracted.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIKinetic parameters of SsNOX38: comparison with other thermophilic NADH oxidasesEnzymeTemperatureSubstrate or cofactork catK mk cat/K mReferences°Cmin −1μmμm −1 · min −1SsNOX3865NADH2654130.64This work65FAD242734.6This workSsNOX3560NADH591619.7(4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar)60FAD5896.788.0(4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar)SaNOX2725NADH21021.010.0(4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar)25FAD2160.6360.0(4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar)TtNOX25NADH3104.1475.0(17.Park H.J. Reiser C.O.A. Kondruweit S. Erdmann H. Schmid R.D. Sprinzl M. Eur. J. Biochem. 1992; 205: 881-885Crossref PubMed Scopus (111) Google Scholar)25FAD31235.88.7(17.Park H.J. Reiser C.O.A. Kondruweit S. Erdmann H. Schmid R.D. Sprinzl M. Eur. J. Biochem. 1992; 205: 881-885Crossref PubMed Scopus (111) Google Scholar)TaNOX25NADH514.839.013.2(18.Cocco D. Rinaldi A. Savini I. Cooper J.M. Bannister J.V. Eur. J. Biochem. 1988; 174: 267-271Crossref PubMed Scopus (45) Google Scholar)25FAD169215.8107.1(2.Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar)CtNOX25NADH19(19.Maeda K. Truscott K. Liu X.L. Scopes R.K. Biochem. J. 1992; 284: 187-190Crossref Scopus (29) Google Scholar)50NADH30(19.Maeda K. Truscott K. Liu X.L. Scopes R.K. Biochem. J. 1992; 284: 187-190Crossref Scopus (29) Google Scholar)k cat was calculated considering theM r of SsNOX38 = 76,000. Open table in a new tab k cat was calculated considering theM r of SsNOX38 = 76,000. The thermophilicity of SsNOX38 was investigated by measuring the NADH oxidase activity at increasing temperatures. As reported in Fig.5, SsNOX38 showed the maximum activity at 87 °C. Above this temperature, inactivation occurred. The data collected in the temperature interval 50–87 °C, analyzed by the Arrhenius equation, gave a straight line (Fig. 5,inset) and a value of 40 kJ/mol was calculated for the activation energy. The heat inactivation of SsNOX38 was evaluated by measuring the residual NADH oxidase activity after heat treatment at two different temperatures (Fig.6 A). At 87 °C the enzyme remained stable for 60 min of treatment, whereas at 105 °C its heat resistance was drastically reduced with a t 12 of about 2 min. The stability of SsNOX38 was also evaluated by ultraviolet-monitored thermal denaturation in the absence or presence of 8 m urea (Fig. 6 B). The corresponding half-denaturation temperatures were 93 and 75 °C, respectively. The stability of the enzyme was also confirmed by its resistance to trypsin. In fact, cleavage of SsNOX38 in two major fragments of about 21 and 14 kDa was obtained after incubation of the protein for 20 h at 37 °C in the presence of trypsin at a 3:1 weight ratio (Fig. 7). The N-terminal amino acid sequence of the 21-kDa fragment M1DEYDIVVIGGGP was identical to that determined on the intact protein, whereas the N-terminal sequence of the first 10 residues determined on the 14-kDa fragment was VANVYVWHEL.Figure 6Heat stability ofSsNOX38. A, heat inactivation ofSsNOX38 was measured at 87 °C (○) or 105 °C (●).B, melting curves of SsNOX38 were measured in the absence (■) or presence (▪) of 8 m urea. See “Experimental Procedures” for details.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Cleavage of SsNOX38 with trypsin. The reaction mixture contained 15 μg ofSsNOX38 in 50 μl of buffer B without phenylmethylsulfonyl fluoride. The reaction was carried out at 37 °C and was started by adding 5 μg of N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-treated trypsin. At timed intervals, 10-μl aliquots were withdrawn, added to 0.25 μg of bovine trypsin inhibitor, and loaded onto a 14% SDS-PAGE. Lane 1, time zero; lane 2, 1 h;lane 3, 4 h; lane 4, 20 h; lane 5, trypsin (1 μg). The positions of the molecular mass standards are shown on theright.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A DNA fragment of about 600 base pairs was synthesized by PCR using as template theS. solfataricus DNA and the nox1 and nox2 primers (see “Experimental Procedures”). This DNA fragment was then cloned in the pGEM-T easy vector and sequenced with the T7 and SP6 primers. The amino acid sequence translated from the nucleotide sequence of this fragment was coincident with the N-terminal sequences of both of the tryptic fragments of purified SsNOX38. Analysis of S. solfataricus DNA by Southern blot, using as probe the32P-labeled PCR (not shown), indicated that a 6.5-kilobase pair EcoRI/PstI fragment contained the entire gene. This fragment was identified by colony hybridization screening of a S. solfataricus library cloned into the pUC18 vector. Fig.7 shows the nucleotide sequence of the gene encoding SsNOX38 and the translated amino acid sequence. The SsNOX38 gene coded for a protein composed of 332 amino acid residues, accounting for a M r 36508, a value close to that determined by SDS-PAGE. The translated primary structure of the enzyme contained the N-terminal amino acid sequence determined on both the purified protein and the 14-kDa tryptic fragment of SsNOX38. The start codon is a GTG triplet (Val), although in the purified protein the N-terminal amino acid detected by Edman's degradation is Met. This feature is common in other S. solfataricus genes (11.De Vendittis E. Bocchini V. Gene. 1996; 176: 27-33Crossref PubMed Scopus (17) Google Scholar). Fig.8 shows the nucleotide sequence of the flanking regions of the SsNOX38 gene also. The 5′-flanking region contains a putative archaeal promoter (20.Thom M. Wich G. Nuleic Acids Res. 1988; 16: 151-163Crossref PubMed Scopus (106) Google Scholar) and a potential Shine-Dalgarno sequence (21.Shine J. Dalgarno L. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1342-1346Crossref PubMed Scopus (2566) Google Scholar), and the 3′-flanking region contains a transcription termination signal. The SsNOX38 gene contains a high content of A and T (34.3 and 27.3%, respectively) as compared with G and C (25.8 and 12.5%, respectively). This finding is in agreement with the selective codon usage in S. solfataricusgenes (11.De Vendittis E. Bocchini V. Gene. 1996; 176: 27-33Crossref PubMed Scopus (17) Google Scholar). The average hydrophobicity and the mean molecular weight of the amino acid residues in the SsNOX38 is slightly lower (4.95 and 110, respectively) than those calculated for other S. solfataricus proteins (5.18 and 114.8, respectively) (22.Dello Russo A. Rullo R. Nitti G. Masullo M. Bocchini V. Biochem. Biophys. Acta. 1997; 1343: 23-30PubMed Google Scholar). SsNOX38 did not show homology with any other NADH oxidase available from the GenBankTM data base. Similarly, it did not show sequence identities with the N-terminal amino acid sequence of the first 20 amino acid residues determined on SsNOX35 andSaNOX27 (4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar). The best alignments were found with thioredoxin reductases and alkyl hydroperoxide reductases. The highest amino acid sequence homology (39.7% identity) was observed with a putative thioredoxin reductase translated from a Bacillus subtilisgenomic DNA fragment (GenBankTM accession number Z93939). Compared with alkyl hydroperoxide reductases, the best alignment was observed with the C-terminal region of the F subunit of alkyl hydroperoxide reductase from Xhantomonas campestris (25% identity; GenBankTM accession number U94336) (Ref. 23.Loprasert S. Atichartpongkun S. Whangsuk W. Mongkolsuk S. J. Bacteriol. 1997; 179: 3944-3949Crossref PubMed Google Scholar and Fig.9). A homodimeric NAD(P)H oxidase with a molecular mass of about 38 kDa/subunit has been isolated from S. solfataricus. The enzyme was different from another NADH oxidase (SsNOX35) previously isolated from the same source (4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar), because the amino acid sequence of SsNOX38 did not contain the N-terminal amino acid sequence of SsNOX35. Nevertheless, theSsNOX35 in its native state is also homodimeric, with one FAD molecule bound per enzyme subunit (4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar). Like all other NADH oxidases, the SsNOX38 is probably involved in vivo in the regeneration of NAD from NADH produced in the aerobic pathway (2.Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar, 24.Higuchi M. Shimada M. Yamamoto Y. Hayashi T. Koga T. Kamio Y. J. Gen. Microbiol. 1993; 139: 2343-2351Crossref PubMed Scopus (112) Google Scholar); the final product of the oxidase reaction is hydrogen peroxide (Fig. 3). SsNOX35 andSaNOX27 also catalyze the transfer of electrons directly from NADH to molecular oxygen to produce hydrogen peroxide. This feature is a distinctive property of thermophilic micro-organisms because, up to now, no H2O-producing NADH oxidase has been isolated from this source. Like SaNOX27 andSsNOX35, SsNOX38 also showed specificity for FAD. In addition, as reported for SsNOX35 (4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar), SsNOX38 is purified also as a flavoenzyme, as deduced from UV and fluorescence spectra (Fig. 2). Despite the two other archaeal enzymes,SsNOX38 can also oxidize β-NADPH (Fig. 4). This feature is shared by the NADH oxidase isolated from Thermus thermophilus (17.Park H.J. Reiser C.O.A. Kondruweit S. Erdmann H. Schmid R.D. Sprinzl M. Eur. J. Biochem. 1992; 205: 881-885Crossref PubMed Scopus (111) Google Scholar). SsNOX38 is very stable to heat inactivation and denaturation (Fig. 6). In fact, at 87 °C the enzyme retains its activity even after a 60-min incubation, whereas the t 12 at 105 °C was 2 min. The heat stability of SsNOX38 was comparable with that reported for SsNOX35 (t 12 = 3.8 min at 105 °C) (3.Masullo M. Ph.D. thesis. Cranfield University, Cranfield, UK1996Google Scholar, 4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar) but higher than that of SaNOX27 (t 12 = 3 min at 100 °C) (3.Masullo M. Ph.D. thesis. Cranfield University, Cranfield, UK1996Google Scholar, 4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar), Thermoanaerobium brokii NOX (t 12 = 30 min at 80–85 °C) (19.Maeda K. Truscott K. Liu X.L. Scopes R.K. Biochem. J. 1992; 284: 187-190Crossref Scopus (29) Google Scholar), andTaNOX (t 12 about 5 min at 87 °C) (2.Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar). This property is also reflected in the thermophilicity ofSsNOX38 (Fig. 5). The highest activity was detected at 87 °C, a temperature about 7 °C higher than that observed for other thermophilic NADH oxidases (4.Masullo M. Raimo G. Dello Russo A. Bocchini V. Bannister J.V. Biotechnol. Appl. Biochem. 1996; 23: 47-54PubMed Google Scholar, 17.Park H.J. Reiser C.O.A. Kondruweit S. Erdmann H. Schmid R.D. Sprinzl M. Eur. J. Biochem. 1992; 205: 881-885Crossref PubMed Scopus (111) Google Scholar). The native FAD-bound SsNOX38 did not show any oxidase activity without additional FAD. This behavior was also observed under anaerobic conditions with the Amphybacillus xylanus NADH oxidase (25.Niimura Y. Yokoyama K. Ohnishi K. Massey V. Biosci. Biotechnol. Biochem. 1994; 58: 2310-2311Crossref Scopus (5) Google Scholar, 26.Ohnishi K. Niimura Y. Yokoyama K. Hidaka M. Masaki H. Uchimura T. Suzuki K. Uozumi T. Kozaki M. Komagata K. Nishino T. J. Biol. Chem. 1994; 269: 31418-31423Abstract Full Text PDF PubMed Google Scholar). In the case of Thermus aquaticus NADH oxidase (2.Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar) it was suggested that the exogenous FAD stimulates the oxidase activity of the enzyme by mediating the electron transfer from the enzyme-bound FAD to molecular oxygen (2.Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar). The same reaction occurs for other related NADH oxidases from A. xylanus (25.Niimura Y. Yokoyama K. Ohnishi K. Massey V. Biosci. Biotechnol. Biochem. 1994; 58: 2310-2311Crossref Scopus (5) Google Scholar, 26.Ohnishi K. Niimura Y. Yokoyama K. Hidaka M. Masaki H. Uchimura T. Suzuki K. Uozumi T. Kozaki M. Komagata K. Nishino T. J. Biol. Chem. 1994; 269: 31418-31423Abstract Full Text PDF PubMed Google Scholar) andSalmonella typhimurium (27.Niimura Y. Poole L.B. Massey V. J. Biol. Chem. 1995; 270: 25645-25650Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Therefore, it is likely that even for SsNOX38 the additional FAD might play a similar role. The K m of SsNOX38 for the β-NADH under aerobic conditions (Table II) was about 7- and 20-fold higher compared with the K m values of theSsNOX35 and SaNOX27, respectively, and at least one order of magnitude higher than K m values reported for TtNOX, TaNOX, and Clostridium thermohydrosulfuricum NOX (CtNOX). TheK m for FAD was comparable with that ofSsNOX35, SaNOX27, and TaNOX but lower than that of NADH oxidase from T. thermophilus. The catalytic efficiency of SsNOX38 was about 15-fold lower than that of the other archaeal NADH oxidases, whereas it was 117-and 21-fold lower than that of thermophilic eubacterial NADH oxidases isolated fromT. thermophilus and T. aquaticus, respectively (Table II and references therein). These findings indicated that archaeal thermophilic NADH oxidases are less efficient compared with their eubacterial counterparts, where their fast aerobic growth is associated with a high NADH consumption (28.Niimura Y. Koh E. Uchimura T Ohara N. Kozaki M. FEMS Microbiol. Lett. 1989; 61: 79-84Crossref Scopus (30) Google Scholar). Many NADH oxidases have been reported as scavenging hydrogen peroxide and therefore fuctioning as peroxidases (2.Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar). They are able to catalyze electron transfer from NADH to several electron acceptors such as methylene blue, cytochrome c, 2,6-dichloroindophenol, and potassium ferricyanide (24.Higuchi M. Shimada M. Yamamoto Y. Hayashi T. Koga T. Kamio Y. J. Gen. Microbiol. 1993; 139: 2343-2351Crossref PubMed Scopus (112) Google Scholar, 17.Park H.J. Reiser C.O.A. Kondruweit S. Erdmann H. Schmid R.D. Sprinzl M. Eur. J. Biochem. 1992; 205: 881-885Crossref PubMed Scopus (111) Google Scholar, 29.Koike K. Kobayashi T. Ito S. Saitoh M. J. Biochem. (Tokyo). 1985; 97: 1279-1288Crossref PubMed Scopus (49) Google Scholar, 30.Saeki Y. Nozaki M. Matsumoto K. J. Biochem. 1985; 98: 1433-1440Crossref PubMed Scopus (55) Google Scholar). In addition, other NADH oxidases have a thiol disulfide oxidase activity mediated by the presence of a redox-active disulfide center constituted of two very close cysteine residues (26.Ohnishi K. Niimura Y. Yokoyama K. Hidaka M. Masaki H. Uchimura T. Suzuki K. Uozumi T. Kozaki M. Komagata K. Nishino T. J. Biol. Chem. 1994; 269: 31418-31423Abstract Full Text PDF PubMed Google Scholar). SsNOX38 is different from both types of NADH oxidases because it did not show any electron transfer activity toward 2,6-dichloroindophenol or DTNB. In fact, as deduced from the sequence of the SsNOX38 gene, no cysteine residues are present in the primary structure of the enzyme. Because, as already suggested for the A. xylanus NADH oxidase (31.Ohnishi K. Niimura Y. Hidaka M. Masaki H. Suzuki K. Uozumi T. Nishino T. J. Biol. Chem. 1995; 270: 5812-5817Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), the reduction of molecular oxygen to hydrogen peroxide did not require the presence of the two cysteines, even for SsNOX38 the mechanism of the reaction might involve the formation of a flavin C/4a-hydroperoxide adduct (32.Entsch B. Ballou D.P. Massey V. J. Biol. Chem. 1976; 251: 2550-2563Abstract Full Text PDF PubMed Google Scholar) followed by the elimination of hydrogen peroxide. The translated amino acid sequence of SsNOX38 (Fig. 8) contains the consensus sequence present in both NAD- and FAD-binding enzymes (33.Wierenga R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (996) Google Scholar). In fact, the ADP-binding βαβ-fold region of many dinucleotide binding enzymes corresponded in the SsNOX38 to the region D5–D36 containing the hydrophobic core of six amino acids (Ile6, Val8, Thr9, Ala22, Ile32, Ala34) and the consensus sequence G10GGPVG. The other putative βαβ-fold region of SsNOX38 corresponds to the segment R152–D183, which includes residues Val153, Ile155, G157GGDSA, Ala166, Leu169, and Ile179. Another consensus sequence conserved among many FAD-binding sites is TXXXXhyhhGD, whereh represents a small hydrophobic amino acid and yis an aromatic residue (34.Eggink G. Engel H. Vriend G. Terpstra P. Witholt B. J. Mol. Biol. 1990; 212: 135-142Crossref PubMed Scopus (214) Google Scholar). This sequence corresponds inSsNOX38 to the segment T271NLPGVYAGGD, thus indicating the presence of an additional binding site for FAD. Furthermore, the consensus sequence GXGXXAXXXAXXXXXXA, typical of the NADP binding proteins (35.Hanukoglu I. Gutfinger T. Eur. J. Biochem. 1989; 180: 479-484Crossref PubMed Scopus (172) Google Scholar), is present also in theSsNOX38. This sequence corresponded to G157GGDSAVDWALTLAPVA, which might explain the oxidase activity of the SsNOX38 toward NADPH. The primary structure of SsNOX38 showed significant sequence homology with thioredoxin reductase and alkyl hydroperoxide reductase from several organisms rather than with other NADH oxidases. The best alignment was found with a putative B. subtilis thioredoxin reductase, homologous to the Escherichia coli thioredoxin reductase (36.Russel M. Model P. J. Biol. Chem. 1988; 263: 9015-9019Abstract Full Text PDF PubMed Google Scholar) and to alkyl hydroperoxide reductase from X. campestris, the latter showing 64% sequence identity with the enzyme from S. typhimurium (37.Tartaglia L.A. Storz G. Brodsky M.H. Lai A. Ames B.N. J. Biol. Chem. 1990; 265: 10535-10540Abstract Full Text PDF PubMed Google Scholar). The multiple alignment showed sequence identities mainly in the regions containing the binding sites for FAD and NAD(P) (Fig. 9). Compared with these enzymes, an important divergence is the lack in SsNOX38 of the redox-active disulfide center. This finding might suggest that theSsNOX38 and NADH oxidoreductase enzymes originated from a common ancestor which, in the course of evolution, underwent different environmental constrains. In conclusion, SsNOX38, like SsNOX35, functions in S. solfataricus cells as a NADH oxidase. However, because the enzyme does not possess reductase activity, its involvement in other possible physiological roles cannot be excluded." @default.
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• W2044903071 title "A NAD(P)H Oxidase Isolated from the Archaeon Sulfolobus solfataricus Is Not Homologous with Another NADH Oxidase Present in the Same Microorganism" @default.
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