A NAD(P)H Oxidase Isolated from the Archaeon Sulfolobus solfataricus Is Not Homologous with Another NADH Oxidase Present in the Same Microorganism

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.

In several bacterial cells, the NADH formed under aerobic conditions by various dehydrogenases is converted to NAD ϩ by NADH oxidase (NOX), 1 which is considered responsible for the maintenance of the intracellular redox balance (1,2). The enzyme has been isolated and characterized from various mesophilic and thermophilic eubacteria (3). There are two types of NADH oxidase; one catalyzes the four-electron reduction of O 2 with formation of H 2 O, and the other catalyzes the two-electron reduction of O 2 to H 2 O 2 . The latter is found in many microor-ganisms including thermophilic eubacteria. We (4) have previously reported the isolation and characterization of NADH oxidase from the archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius. The S. solfataricus enzyme is a homodimer composed of two subunits of M 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 H 2 O 2 -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). In contrast, the N-terminal sequence of the first 23 amino acid residues of SaNOX27 showed 57% identity with a ␤-alanine-piruvate aminotransferase (5) and 47% identity with a putative NADP reductase from the archaeon Methanococcus jannaschii (6).
In this work, we report the purification, the biochemical characterization, and the cloning of the gene of a second H 2 O 2forming NAD(P)H oxidase from the archaeon S. solfataricus (SsNOX38). The relationship of the primary structure of this enzyme with that of other NADH oxidases and oxidoreductases is also analyzed.

Methods
Enzymatic Assays-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 (⑀ ϭ 6220 M Ϫ1 ⅐cm Ϫ1 ). One unit of enzyme was defined as the amount of enzyme that catalyzes the oxidation of 1 mol 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). The reaction was carried out in 0. 25  Assays for the DTNB and 2,6-dichloroindophenol reductase activities of NADH oxidase were performed as already described (2). The reaction mixture contained 0.3 mM EDTA, 75 M 2,6-dichloroindophenol or 0.4 mM DTNB, and 1 mM NADH in 1 ml final volume of 50 mM phosphate buffer, pH 7.0, The reaction was started by adding 3 g of purified SsNOX38. The absorbance was monitored at 600 nm for the 2,6-dichloroindophenol and at 412 nm for DTNB.
Purification of SsNOX38 -S. solfataricus cells (strain MT-4, ATCC 49255) were grown as already reported (9). 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 ϫ 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 ϫ 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-l 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 ϫ 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-l 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 ϫ 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-l 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 Ϫ20°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 g/ml final concentration) and finally analyzed on 14% SDS-PAGE.
Kinetic Parameters of SsNOX38 -K m and V 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 and V were calculated by Lineweaver-Burk plots.
Heat Stability and Thermophilicity of SsNOX38 -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 plotted versus temperature (10). The thermophilicity of SsNOX38 was evaluated in the temperature range of 50 -100°C. At each temperature the reaction was followed spectrophotometrically as described above.
Cloning of the SsNOX38 Encoding Gene-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, ATG⅐GAT⅐GGA⅐TAT⅐GAT⅐ATA⅐GT (nox1), was deduced from the Nterminal sequence MDEYDIV of the purified protein, whereas the reverse primer, TC⅐ATG⅐CCA⅐TAC⅐ATA⅐TAC⅐ATT (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). A S. solfataricus DNA library was prepared into the pUC18 cloning vector, upon digestion of both DNAs with EcoRI/PstI restriction enzymes. About 2 ϫ 10 3 colonies were analyzed using as probe the 32 P-labeled PCR fragment.
Other Methods-Electrophoretic analysis was performed on 14% polyacrylamide gels in the presence of SDS (12) using appropriate M r standards. Protein concentration was determined according to Bradford (13). Nucleotide sequencing of the SsNOX38 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). 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 GenBank TM data base using the BLASTP program (15). Sequence analyses and alignments were established with the help of a nucleic acid and protein analysis software system packed with the CLUSTAL program (16).

RESULTS
Purification of SsNOX38 -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), 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.
Molecular Properties of SsNOX38 -The molecular mass of SsNOX38 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. 2A). The fluorescence spectrum showed a maximum at about 520 nm (Fig. 2B). These features suggest that, like SsNOX35 (4), even purified SsNOX38 is bound to a flavin cofactor.
Substrate Specificity and Kinetic Properties of SsNOX38 -SsNOX38 catalyzed the oxidation of NADH in the presence of molecular oxygen with formation of hydrogen peroxide. A stoichiometric amount of H 2 O 2 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 ␣-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, 17-19). 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. Thermophilicity and Stability of SsNOX38-The thermophilicity of SsNOX38 was investigated by measuring the NADH oxidase activity at increasing temperatures. As reported in Fig. 5, Ss-NOX38 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. 6A). At 87°C the enzyme remained stable for 60 min of treatment, whereas at 105°C its heat resistance was drastically reduced with a t1 ⁄2 of about 2 min. The stability of SsNOX38 was also evaluated by ultravioletmonitored thermal denaturation in the absence or presence of 8 M urea (Fig. 6B). 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.
Cloning and Sequencing of the SsNOX38 Gene-A DNA fragment of about 600 base pairs was synthesized by PCR using as template the S. 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 the 32 P-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 struc-ture 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). Fig. 8 shows the nucleotide sequence of the flanking regions of the SsNOX38 gene also. The 5Ј-flanking region contains a putative archaeal promoter (20) and a potential Shine-Dalgarno sequence (21), and the 3Ј-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. solfataricus genes (11). 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).
Amino Acid Sequence Comparison-SsNOX38 did not show homology with any other NADH oxidase available from the GenBank TM 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 and SaNOX27 (4). 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 subtilis genomic DNA fragment (GenBank TM 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; GenBank TM accession number U94336) (Ref. 23 and Fig. 9). DISCUSSION 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 (Ss-NOX35) previously isolated from the same source (4), because the amino acid sequence of SsNOX38 did not contain the Nterminal amino acid sequence of SsNOX35. Nevertheless, the SsNOX35 in its native state is also homodimeric, with one FAD molecule bound per enzyme subunit (4).
Like all other NADH oxidases, the SsNOX38 is probably involved in vivo in the regeneration of NAD from NADH produced in the aerobic pathway (2,24); the final product of the oxidase reaction is hydrogen peroxide (Fig. 3). SsNOX35 and SaNOX27 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 H 2 O-producing NADH oxidase has  been isolated from this source. Like SaNOX27 and SsNOX35, SsNOX38 also showed specificity for FAD. In addition, as reported for SsNOX35 (4), 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 ␤-NADPH (Fig. 4). This feature is shared by the NADH oxidase isolated from Thermus thermophilus (17).
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 (25,26). In the case of Thermus aquaticus NADH oxidase (2) 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). The same reaction occurs for other related NADH oxidases from A. xylanus (25,26) and Salmonella typhimurium (27). Therefore, it is likely that even for SsNOX38 the addi-