NADH oxidases (NOXs) catalysing the oxidation of NADH to yield NAD+ and H2O, H2O2, or both play an important role in protecting organisms from oxidative stress and maintaining the balance of NAD+/NADH. A gene encoding NOX was identified from Methanobrevibacter smithii (NOX-ms), the predominant archaeon in the human gut ecosystem. Subsequent analyses showed that it is an FAD-containing protein with a subunit molecular mass of 48 kDa. NOX-ms was purified to homogeneity after expression in Escherichia coli. NOX-ms catalysed the oxidization of NADH and converted O2 to H2O with an optimal pH of 7.5 and a temperature optimum of approximately 37°C. The Vmax and Km values were 42.6–44.1 unit/mg and 47.8–54.6 μM for NADH. The apparent Vmax and Km for oxygen were 189.5–196.1 unit/mg and 14.6–16.8 μM. The mutation analysis suggests that Cys42 in NOX-ms plays a key role in the four-electron reduction of O2 to H2O. Quantitative reverse transcription-PCR (RT-qPCR) revealed that transcription of NOX-ms was also up-regulated after exposing the cells to oxidative stress and glucose. Finally, the potential of NOX-ms as a target to control colonization of M. smithii and its possible applications are discussed.
Recently, a role of the gut microbiota has been attracting more attention. Besides the many bacterial species, the human gut contains several archaeal species but only three distinct species within the group of methanogenic archaea have been isolated from human faeces, Methanobrevibacter smithii , Methanosphaera stadtmanae  and Methanomassilicoccus luminyesis . M. smithii belonging to the Methanobacteriaceae family is the dominant archaeon in the human gut ecosystem . In children from 1 to 10 years of age, the prevalence of M. smithii has been found in 88% . This organism plays an important role in the efficient digestion of polysaccharides (complex sugars) by consuming the end products of bacterial fermentation . Metagenomics studies of the gut microbial communities in genetically obese mice have shown that M. smithii exhibited an enhanced expression of the genes involved in polysaccharide degradation and possess a greater capacity to promote adiposity when transplanted into germ-free recipients . A recent research showed that M. smithii colonization was associated with an increased risk of overweight children from 6 to 10 years of age . M. smithii may thus be a therapeutic target for childhood overweight and obesity by reducing energy harvesting.
NADH oxidase (NOX) is a member of the flavoprotein disulfide reductase family that catalyses the pyridine-nucleotide-dependent reduction of various substrates, including O2, H2O2 and thioredoxin . There are two types of NOXs that are H2O2-forming (NOX-1) and H2O-forming (NOX-2) respectively. NOX-1 catalyses the two-electron reduction of O2 to H2O2 by NADH, whereas NOX-2 catalyses the four-electron reduction of O2 to H2O by NADH . The deduced amino acid sequences between the NOX-1 and NOX-2 showed low homology [9,10]. NOXs play diverse physiological roles, depending on its substrates and products in different organisms. NOX-1 is part of an alkyl hydroperoxide reductase system in vivo in combination with alkyl hydroperoxide reductase subunit C in Amphibacillus xylanus and Streptococcus mutans [11,12]. NOX-1 from thermophilic Archaeoglobus fulgidus may be involved in electron transfer in sulfate respiration . NOX-2 are considered to be important enzymes in protecting against oxidative stress through their capacity to reduce O2 to H2O without the formation of harmful reactive oxygen species  and in regenerating NAD+ during aerobic mannitol metabolism, acts an important role in aerobic energy metabolism in O2-tolerant Streptococcus mutans and maintaining the balance of NAD+/NADH . In application, some of the NOX-2 were successfully applied to control the level of intracellular cofactors to redirect cellular metabolism [15–18].
Despite the importance of NOX in protecting against oxidative stress and energy metabolism, little is known about the function of NOX in M. smithii. In the present study, a water-forming NOX was identified from M. smithii. The recombinant His-tag NOX from M. smithii (NOX-ms) was efficiently produced in a bacterial expression system and purified by immobilized metal affinity chromatography. Afterward, the enzyme was biochemically characterized and used mutants to analyse the catalytic mechanism. The expression level of NOX-ms under different conditions was finally analysed.
MATERIALS AND METHODS
Protein expression and purification
M. smithii strain PS (ATCC 35061) was cultivated in 125 ml serum bottles containing 15 ml of Methanobrevibacter complex medium supplemented with 3 g/l formate, 3 g/l acetate and 0.3 ml of a freshly prepared, anaerobic, filter-sterilized 2.5% Na2S solution. The remaining volume in the bottle (headspace) contained a 4:1 mixture of H2 and CO2; the headspace was replenished every 1–2 d during a 6-d growth period at 37°C. DNA was recovered from harvested cell pellets using the Qiagen Genomic DNA Isolation kit, with mutanolysin (1 unit/mg wet-weight cell pellet; Sigma) added to facilitate microbe lysis.
M. smithii genomic DNA was used as a template in a PCR, which isolated NOX-ms (Msm_0046, WP_004033913) using the following oligonucleotide primers: forward, 5′-CG G AATTC ATG AAA GTT GTT ATT G-3′ and reverse, 5′-CCG CTCGAG TTA GTT AAA TTT CTT AC-3′. The primers introduce restriction sites EcoRI and XhoI (underlined) respectively. PCR products were ligated into the pET28 (a) vector and sequenced before transformation into BL21 (DE3). Escherichia coli BL21 (DE3) cells containing the pET28-NOX-ms plasmid were cultured. When the A600 reached 0.7, IPTG was added to induce protein expression. The cells were cultured in the presence of IPTG for 4 h with shaking and then harvested and resuspended in lysis buffer containing 50 mM Tris (pH 8.0), 300 mM NaCl, 20 mM 2-mercaptoethanol and 20 mM imidazole. The cell suspension was sonicated and centrifuged at 20000 g for min, and the supernatant was loaded on a Ni-NTA column. After washing the column with lysis buffer, NOX-ms was eluted using an imidazole gradient (50–250 mM). Purified protein was separated on a SDS/10% PAGE and visualized. Protein concentrations were estimated using the Bradford method and BSA as a standard .
Site-directed mutagenesis of NOX-ms
The primers used for the single cysteine to serine mutant (underlined) were as follows: Cys42, forward, 5′-TAT TCT CCA GCT GCT ATT CCT-3′; reverse, 5′-AGG AAT AGC AGC TGG AGA ATA-3′; Cys230, forward, 5′-GAC GGA AGC GCT ATT GAT GCA-3′; reverse, 5′-TGC ATC AAT AGC GCT TCC GTC-3′. The pET28a-NOX-ms plasmid was used as the DNA template. The PCR reaction was performed for 18 cycles (94°C for 30 s, 55°C for 1 min and 68°C for 12 min). After amplification, the PCR mixture was digested with DpnI and used to transform E. coli BL21(DE3). The mutant was confirmed by DNA sequencing. The NOX-ms-C42A and NOX-ms-C230A were purified by the same methods as that of the wild-type protein, as described above.
Spectra analysis of NOX-ms and Apo-NOX-ms preparation
The purified enzymes were scanned in the air-saturated 100 mM sodium phosphate buffer (pH 7.0) at room temperature in a 1.0 ml quartz cuvette. The absorption spectrum (300–800 nm) was recorded in the spectrophotometer.
The purified NOX-ms from E. coli is a holoenzyme with FAD. The protein was dialysed with 100 mM phosphate buffer (pH 7.2) containing 2.4 M (NH4)2SO4, 20 mM 2-mercaptoethanol and 0.5 mM EDTA, and then loaded on the hydrophobic interaction chromatography column equilibrated with the same buffer. FAD was eluted with equilibration buffer saturated with NaBr (pH 3.5). The column was balanced again with the equilibration buffer, and the apoprotein was eluted with 100 mM phosphate buffer.
Assays for NOX-ms activity
The NOX activity of the recombinant protein was examined by time-dependent removal of NADH in aerobic conditions. The assays were performed in 50 mM sodium phosphate buffer (pH 7.2), 0.5 mM NADH or NADPH and 100 mM NaCl at the indicated temperatures. The reaction was started by adding NOX-ms in the amounts indicated. The rate of NADH consumption was measured by monitoring the decrease in A340 in a UV–visible spectrophotometer (INESA Instrument). One unit of activity was defined as the amount of enzyme catalysing the oxidation of 1 μmol NADH or O2 per min at 37°C in 50 mM sodium phosphate buffer (pH 7.2).
For kinetic studies, the initial velocities of the enzymatic reaction were examined by varying the concentration of NADH (from 0.02 to 0.6 mM) in the optimal conditions. For the O2 monitor assay, NOX activity was measured using an anaerobic glass cuvette in 50 mM potassium phosphate buffer (pH 7.0) inflating with different concentration of oxygen. The reaction was started by the addition of enzyme solution, and the decrease in oxygen concentration was monitored with a Clark-type oxygen electrode (Rex Electric, JPB-607A). Values of the Michaelis constants (Km) and maximal velocity (Vmax) were obtained by mathematical calculations according to SigmaPlot software. The parameters were determined by three separate experiments.
Quantification of H2O2
NOX assay mixtures containing various concentrations of NADH (0.1–0.5 mM) were incubated at 37°C and the reaction was allowed to go to completion. The solution (250 μl) was combined with 50 μl of a solution containing Amples Red and 100 unit/ml horseradish peroxidase (Aladdin). After incubation at room temperature for 30 min, the absorbance at 560 nm was measured. The amount of H2O2 produced in the assay was determined according to the absorbance value and a standard curve.
Quantitative reverse transcription-PCR)
The culture of M. smithii was carried out in as described above. The cells were shocked by exposure to oxygen (1–8% O2 was sparged into the growth environment) or in the presence of glucose (1–4 g/l) and incubating for 6 h. Survival of the cells was estimated by the three-tube most probable number method after exposure to stress. The cells were harvested and RNA was prepared with TRIzol reagent (Invitrogen). To ensure complete removal of any contaminating DNA, all RNA preparations were given a DNAase treatment (Thermo Scientific Fermentas). RNA was quantified with a spectrophotometer and cDNA was synthesized with the Universal RiboClone cDNA Synthesis System (Promega) according to the manufacturer's protocols. The reaction products were serially diluted to find the adequate concentration for real-time PCR analysis using the following primers: 5′-GGT GAC GGA AGC TGT ATT GA-3′ and 5′-AGC CCA TCT TCC GAT ATC AC-3′. Real-time PCR was performed in CFX96 Real-Time PCR System (Bio–Rad Laboratories), using SYBR Green PCR Master Mix (Toyobo, Japan). The relative fold changes were determined from cycle threshold (CT) values using the ΔΔCT method. The reactions for detection of 16S rRNA (Msm_1801) levels were used for normalization between the different samples, which were amplified with primers: 5′-CTG CAG CTT AAC TGT GGG AA-3′ and 5′-GGT CCT CCC AGG ATT ACA GA-3′. The experiments were analysed in three independent assays, with at least three technical replicates included in each PCR to ensure reproducibility.
Sequence analysis of NOX-ms
Homology and domain search of the M. smithii genome identified a homolog of NOX (NOX-ms, Msm_0046) that was not characterized before. Sequence analysis revealed that nox-ms encodes a protein of 444 amino acids, with a theoretical pI of 4.8 and a theoretical molecular mass of 48.180. A BLAST-P search in National Center for Biotechnology Information (NCBI) for the NOX-ms sequence revealed the most significant homology (35–64% identity) with the proteins from methanogenic archaea (Methanothermobacter thermoautotrophicus, Methanocaldococcus jannaschii). NOX-ms also showed >30% identity to the proteins from other archaea (Thermococcus kodakarensis, Pyrococcus abyssi), bacteria (Halonatronum saccharophilum, Hydrogenibacillus schlegelii etc.) and eukaryotes (Cladophialophora immunda, Exophiala oligosperma, Metarhizium anisopliae, etc.). To explore the evolutionary relationship between NOX-ms and other annotated NOXs, the MEGA6 program was used to construct a phylogenetic tree from amino acid sequence data. Although the bootstrap values were somewhat low because of a large number of sequences, more significant bootstrap values in the distal branches allowed us to infer those proteins from similar species that were derived from a common ancestor. Furthermore, their positions in the dendrogram were independent of the method used for phylogenetic reconstruction (results not shown).
The amino acid composition of NOX-ms revealed the presence of eight cysteine residues and multiple sequence alignment (Figure 1B) revealed that Cys42 is located at a similar position to that of the cysteine residue in the conserved active site of other NOXs. The conserved motifs were also identified in the amino acids sequence alignment. One was an FAD-binding domain containing the AMP-binding and FMN-binding motifs observed in enzymes belonging to the glutathione reductase family. The other domain was a glycine-rich NAD-binding motif located between the AMP-binding and FMN-binding motifs (two FAD-binding domains) .
Phylogenetic and sequence analysis of NOX-ms and other NOXs
Purification of NOX-ms and apo-NOX-ms preparation
In order to understand the function of NOX-ms, the nox-ms gene was successfully amplified from genomic M. smithii DNA and cloned to plasmids adding a His-Tag at' the N-terminus. The purification of recombinant NOX-ms from E. coli was performed by affinity chromatography as described in section ‘Materials and methods’. SDS/PAGE analysis of recombinant NOX-ms revealed a molecular mass of approximately 50 kDa (Figure 2A). Meanwhile, the enzymes with N-terminal His-Tag were soluble and could be purified as a yellowish solution due to the bound FAD, because the isoalloxazine ring system in FAD can induce light absorbance in the UV and visible spectral range, giving rise to the yellow appearance of flavin and flavoproteins. Purified NOX-ms from E. coli has absorption maxima at 378 nm and 456 nm, with a shoulder at 480 nm, which is characteristic spectral feature of proteins with bound flavin cofactors. As NOX-ms contains FAD as a prosthetic group, apo-NOX-ms was prepared by hydrophobic interaction chromatography under acidic conditions (pH 3.5) with saturated NaBr buffer , in order to confirm the function of FAD. The absorption spectrum of apo-NOX-ms did not show any significant absorbance in the visible region, revealing that FAD was indeed absent (Figure 2B). The holoenzyme can be reconstituted by incubating equimolar concentrations of apomonomers and FAD at room temperature for 5 min. After dialysis to remove unbinding FAD, the absorbance of the enzyme can be recovered. Compared the absorbance of the native enzyme and reconstituted enzyme, the absence of an absorbance feature in the 480 nm region of the reconstituted enzyme were observed. A similar lack of absorbance shoulder in this region is seen in spectra reported in previous studies of NOX  and other flavoproteins , implicating that the protein may have lower affinity to cofactors than the native protein .
Purification and spectra of NOX-ms
Catalytic characterization of NOX-ms
NOXs from Lactobacillus sanfranciscensis , Pyrococcus horikoshii  and Thermococcus profundus  accept both NADH and NADPH as cofactors. The NOX and NADPH oxidase activity of NOX-ms was also measured. The assays revealed that NOX-ms preferred NADH as the active substrate, and the activity towards NADPH was approximately 20% of that to NADH (Supplementary Figure S1). In the further research, NADH was used as the reducing substrate to determine the function of NOX-ms. In the temperature activity experiments, the NOX activity of NOX-ms was measured at temperatures ranging from 25°C–50°C at a constant pH of 7.2. The results revealed that the optimum temperature for the enzyme was approximately 37°C (Figure 3A). To study the effect of pH, the NOX activity of the purified enzyme were surveyed between pH values of 5.0 and 10.0 at an assay temperature of 37°C. The pH profile of the purified NOX-ms is shown in Figure 3(B). The enzyme was most active between pH values 6.5–8.0, with maximal activity at pH 7.5. The kinetics of recombinant NOX-ms were analysed using NADH as a substrate by varying its concentration. The Michaelis–Menten equation was used to calculate the kinetic parameters (Figure 3B). NOX-ms could catalyse NADH oxidization with an apparent Km=47.8–54.6 μM and Vmax=42.6–44.1 unit/mg (n=9). The kinetic parameters for O2 was also measured by varying O2 concentration and keeping NADH concentration to be constant, which showed that NOX-ms could remove O2 with an apparent Km=14.6–16.8 μM and Vmax=189.5–196.1 unit/mg (n=9).
Enzyme activity assays of NOX-ms
To determine whether flavin cofactors were required for the enzymatic activity of NOX-ms, FMN, FAD and riboflavin were added as equimolar concentrations of apomonomers of holo-NOX-ms and the activity was measured. The results showed that external flavin cofactors have no significant effect on the activity (Supplementary Figure S2A). To further confirm the function of flavin cofactors, holo-enzyme and apo-enzyme activities were assayed. The activity of the reconstituted enzyme by FAD was also measured. These assays revealed that FAD significantly restored the oxidase activity of apo-NOX-ms (Figure 3D). Addition of other flavin cofactors such as FMN and riboflavin to apo-enzyme could not recover the enzyme activity. These results clearly indicated that NOX-ms is an FAD-dependent NOX.
Previous study showed that cysteine may function as the non-flavin redox centre in NOX . The sequence alignment of NOXs (Figure 1B) revealed that NOX-ms contains eight cysteine residues and Cys42 may be the active site. As cysteines are important residues for NOX enzyme activity, we replaced Cys42 with alanine to analyse the function of the two residues. Cys230 was also mutated as a control. After purification using the same method as that used for the wild-type enzyme, NOX assays were performed with the two mutants under the same conditions as used for the wild-type. The results showed that the C230A mutant had similar NOX activity to that of the wild-type protein; however, the C42A mutant had <10% of the NOX activity of the wild-type protein (Figure 3D). Considering these results, Cys42 may provide the essential second redox centre in addition to the flavin.
Due to a conserved cysteine residue at position 42, NOX-ms should be an H2O-forming NOX. In order to determine the product of the NOX activity of NOX-ms, H2O2 was quantified using the Amples Red and horseradish peroxidase method . The results demonstrated that less than 1% of the theoretical yield of hydrogen peroxide could be detected.
The expression of
As NOX may have a key role in oxidative stress tolerance  and sugar metabolism , gene expression level was studied by quantitative reverse transcription-PCR (RT-qPCR) to investigate the physiological role of NOX-ms. RNA was isolated from M. smithii cells grown under different concentration of oxygen. The results showed that there were no significant differences in the frequency of viable cells compared with control (Supplementary Figure S1). The relative expression patterns of nox-ms are shown in Figure 4. The lowest transcription level could be seen under anaerobic conditions. With increase in oxygen concentration, the expression level increased. Expression of nox-ms was also up-regulated by glucose in the media. Therefore, the physiological function of NOX-ms seemed to be related to both oxidative stress tolerance and sugar metabolism.
Expression level of
nox-ms gene under oxidative stress and in the presence of glucose
M. smithii is the leading representative species in healthy volunteers  and plays a central role in the regulation of gut redox . NOX is the key enzyme maintaining the redox balance in bacteria, plants and mammalians [30–32]. In addition, the biotechnological applications of NOXs as the regenerating system in redox reactions have been recently highlighted . By the potential importance of the enzyme in M. smithii, a deeper understanding of its function is highly desirable. In the present study, we have demonstrated NOX-ms can efficiently reduce O2 to produce H2O using NADH as an electron donor. In addition, the activity assays of the wild-type, apo-enzyme and mutants showed that NOX-ms is an FAD-dependent enzyme and Cys42 is the active site residue. These results indicate that both FAD and Cys42 participate in the direct four-electron transfer reduction of O2 to H2O. Furthermore, the transcription level of NOX-ms was up-regulated under aerobic conditions and by glucose. Considering NOX-ms is involved in sugar metabolism and the relationship between M. smithii and overweight, we propose that NOX-ms can be a potential target to control the colonization of M. smithii.
H2O-forming NOXs in facultative anaerobes play an important role in the oxygen tolerance of these bacteria [8,34]. For example, mutants of Streptococcus pyogenes (facultative anaerobe) and Brachyspira hyodysenteriae (an aerotolerant anaerobe) with H2O-forming NOX-deficiency are unable to grow under high-O2 conditions, indicating the importance of NOX-scavenging activity against harmful O2 [34,35]. In Clostridium aminovalericum, an obligate anaerobe, H2O-forming NOX also exists as an oxygen-detoxifying enzyme . In archaea, NOX from Pyrococcus furiosus produces both H2O2 (77%) and H2O (23%)  and NOX from T. profundus can catalyse electron transfer from NADH and NADPH to O2 and predominantly produce H2O . NOX-ms only made H2O during its reaction with NADH and O2. The Vmax value to O2 is approximately four times higher than that to NADH. The Km to O2 is much lower than the value to NADH, implicating NOX-ms has much higher affinity to O2. The Km value to O2 is also lower than the value (61.9 μM) of the enzyme from C. aminovalericum . Meanwhile, the expression of NOX-ms was up-regulated by oxygen. We, therefore, propose that NOX-ms may decompose O2 and protect M. smithii from oxidative stress.
Lots of the oxidation reactions need the expensive cofactor, NAD+. At present, several processes have been applied to overcome cofactor regeneration, such as co-immobilization of bi-enzyme cascades on porous supports , engineering of glycerol dehydrogenase for regeneration of N6-CM-NAD+  and simultaneous expression of two or more enzymes in a single cell . One promising approach to regenerate NAD+ pools is the use of NOXs that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+ . Several NOXs from Thermus thermophiles , Lactobacillus pentosus  and Bacillus subtilis  have been reported to be used as the cofactor regeneration enzymes. On the other hand, NOX-ms was up-regulated by glucose, implicating that NOX-ms would utilize the glycolytic NADH to produce NAD+. Considering the advantage of producing water, NOX-ms has also the potential application in NAD+ regeneration.
Mingguang Yan performed the experiments and collected the data. Weibing Yin and Xiao Fang analysed the data. Jianjun Guo contributed materials and reagents. Hong Shi wrote the manuscript.
This work was supported by The First People's Hospital of Shangqiu City.