Cloning and Characterization of Filamentous Fungal S-Nitrosoglutathione Reductase from Aspergillus nidulans

Abstract

S-Nitrosoglutathione reductase (GSNOR) metabolizes S-nitrosoglutathione (GSNO) and has been shown to play important roles in regulating cellular signaling and formulating host defense by modulating intracellular nitric oxide levels. The enzyme has been found in bacterial, yeast, mushroom, plant, and mammalian cells. However, to date, there is still no evidence of its occurrence in filamentous fungi. In this study, we cloned and investigated a GSNOR-like enzyme from the filamentous fungus Aspergillus nidulans. The enzyme occurred in native form as a homodimer and exhibited low thermal stability. GSNO was an ideal substrate for the enzyme. The apparent K_m and k_cat values were 0.55 mM and 34,100 min^-1, respectively. Substrate binding sites and catalytic center amino acid residues based on those from known GSNORs were conserved in this enzyme, and the corresponding roles were verified using site-directed mutagenesis. Therefore, we demonstrated the presence of GSNOR in a filamentous fungus for the first time.

Introduction

Nitric oxide (NO) is ubiquitous in the biological sphere. The main source of NO in vertebrate systems is enzymatic oxidation of L-arginine to L-citrulline by nitric oxide synthase (NOS) [21,32]. Some microbial denitrification and nitrification processes also generate NO as a byproduct [11]. NO is reported to have diverse physiological and pathophysiological roles, such as cellular signal transduction and host defense in biological systems [22,30]. As a biological signaling molecule, NO is actually not stable enough to regulate numerous physiological processes owing to its short half-life [23]. For this reason, cells employ S-nitrosoglutathione (GSNO), a product of nitrosation, covalently binding NO to the cysteine thiol of intracellular glutathione (GSH), allowing the cell to store and transport NO in this bound form. GSNO spontaneously transfers NO to the cysteine residues of cysteine-containing proteins to form nitrosated proteins (protein-SNOs) for cell signaling [12]. As an innate immune molecule, NO is a vital component of the host defense system in animals. NO can inhibit or kill a broad range of microorganisms by blocking essential microbial physiological processes, including respiration and DNA replication [6,35]. Once microorganisms are subjected to nitrosative stress (a deleterious effect caused by NO and NO donors), the abundant intracellular GSH could act as a buffer pool for NO, producing GSNO to alleviate the abrupt shock from NO [2]. The resulting GSNO will subsequently be enzymatically decomposed [27,31]. The formation and decomposition of GSNO has been regarded as a strategy for the prevention of NO toxicity. Thus, endogenous GSNO acts as a cell-signaling molecule as well as a NO-bearing species.

GSNO decomposition is the process of denitrosation, or removing the NO group, of the thiol of GSNO. It is a key regulated event in both cellular signal transduction and host defense. There are two enzyme systems that operate as efficient GSNO denitrosases: the S-nitrosoglutathione reductase (GSNOR) system, which comprises GSH and GSNOR, and the thioredoxin (Trx) system, which comprises Trx and Trx reductase (TrxR). GSNOR specifically breaks down endogenous and exogenous GSNO to yield glutathione disulfide (GSSG), NH4+, hydroxylamine, and other unknown products in a NADH-dependent manner [15]. The Trx system can denitrosate GSNO, releasing GSH and nitroxyl (HNO) in vitro [31], but whether the same reaction occurrs in vivo remains unknown. Besides GSNOR and Trx systems, GSNO can also be metabolized by other previously known enzymes, such as xanthine oxidase, protein disulfide isomerase, superoxide dismutase, glutathione peroxidase, nitroreductase, and carbonyl reductase [3,13,16,17,28,33]. However, to date, among the abovementioned candidate enzymes, only GSNOR has been verified as the actual physiological GSNO denitrosase.

Filamentous ascomycete fungi can persist in high NO concentrations and are of pharmaceutical and medical importance. Aspergillus nidulans and Aspergillus oryzae are powerful examples of pathogenic Aspergillus species to study the nitrosative stress protection mechanism. From them, Zhou et al. [37-40] previously identified flavohemoglobin [39,40], NO2- reductase, porphobilinogen deaminase [38], and NO-inducible nitrosothionein [37] as fungal NO-tolerating enzymes.

GSNORs have been reported in widespread species, including bacterial, yeast, mushroom, plant, and mammalian cells, and their biochemical properties and physiological roles have been described in detail [10,18,20,24,25]. Although Zhou et al. [37] recently identified a GSNOR-like gene in A. nidulans, there is a lack of research evaluating the corresponding information of filamentous fungal GSNOR. In the present study, we analyzed the full-length cDNA sequence of a GSNOR-like gene from A. nidulans, and biochemically characterized the recombinant fungal enzyme. Accordingly, we corroborated the presence of GSNOR in a filamentous fungus and evaluated its potential physiological functions.

 

Materials and Methods

Cloning of cDNA and Expression Vector Construction

Aspergillus nidulans strain ABPU1 (biA1; pyrG89; wA3; argB2; pyroA4) was a gift from Hiroyuki Horiuchi (University of Tokyo, Tokyo, Japan). The strain was grown at 37℃ in minimal medium (10 mM NaNO3, 10 mM KH2PO4, 2 mM MgSO4, 7 mM KCl, 1% glucose, and 2 ml/l Hunter’s trace metals) supplemented with 0.4 mg/l biotin and 0.4 mg/l pyridoxine. Total RNA was extracted from A. nidulans cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The full-length cDNA of Aspergillus nidulans GSNOR was obtained by 5’-3’ RACE using the GeneRacer Kit (Invitrogen), following the protocol suggested by the supplier. To obtain 5’ ends, cDNA was amplified using the following primers: GSNOR reverse gene-specific primer 5’-CTCTCACCCCAGCCCTTGTG-3’, GSNOR reverse gene-specific nested primer 5’-CGCCAACGTTTCCGGTGCAG-3’, GeneRacer 5’ primer, and GeneRacer 5’ nested primer. To obtain 3’ ends, cDNA was amplified using the following primers: AnGSNOR forward gene-specific primer 5’-CGGTATCACGACCGGCTACG-3’, AnGSNOR forward gene specific nested primer 5’-GAGGAGGGCTCCAACATCGC-3’, GeneRacer 3’ primer, and GeneRacer 3’ nested primer. Amplified 5’- and 3’-RACE DNA was cloned using pT7Blue T-vector (Novagen, Madison, WI, USA) and then sequenced separately.

Expression and Purification of the Recombinant GSNOR

The AnGSNOR gene was subcloned using the forward primer 5’-CTGCGCATATGGCTAGCACTGTTGGCAAG-3’ (NdeI restriction site underlined), and the reverse primer 5’-GGCTGAAGCTTATGACAGGTCAACAACGC-3’ (HindIII restriction site underlined). The PCR-amplified product was digested with the corresponding restriction enzymes and inserted into pET28b (+) (Novagen). E. coli BL21 (DE3) containing the vector was cultured at 37℃ in the presence of 50 μg/ml kanamycin to an OD of 0.5. The culture was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside and then incubated overnight at 30℃. When the culture ended, the cells were harvested and then disrupted by a high pressure homogenizer (JNBIO, Guangzhou, China). The supernatant was collected by centrifugation at 10,000 ×g for 10min and then applied to affinity chromatography with HiTrap Chelating HP (GE Healthcare), which was attached to an AKTA purifier (GE Healthcare). The column was washed with 20 mM sodium phosphate buffer (pH 7.2) and 10 mM imidazole, and the bound proteins were eluted with a gradient of 10–500 mM imidazole.

Molecular Mass Determination

The purified enzyme sample was applied to a Superose 6 HR 10/30 column (GE Healthcare) and then eluted with 20 mM sodium phosphate buffer with 150 mM NaCl at a flow rate of 0.5 ml/min. The absorbance of the effluent was recorded at 280 nm. The molecular mass of the enzyme was calculated from the mobilities of the following standard proteins: thyroglobulin (660 kDa), glutamate dehydrogenase (290 kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), adenylate kinase (32 kDa), and cytochrome c (12.4 kDa). SDS-PAGE (12% gel) was also performed to estimate the molecular mass of the purified proteins.

Enzyme Assay

The GSNO reductase activity and aldehyde and alcohol dehydrogenase activity of AnGSNOR were both detected at 37℃ by monitoring the consumption or formation of NADH at 340 nm, respectively. GSNO reductase activity was determined in 1 ml of 100 mM sodium phosphate buffer (pH 6.0) containing 400 μM NADH, 400 μM GSNO, and 1.6 μg of purified enzyme. Dehydrogenase activity was determined in the same buffer containing 1 mM NAD+, 1 mM GSH, and 1 mM each substrate (formaldehyde, acetaldehyde, propionaldehyde, and some alcohols). NADH and NAD+ were always prepared freshly. All absorbance spectra were recorded with a U-5100 spectrophotometer (Hitachi, Japan). The initial reaction velocity was measured from the linear part of the curve. The apparent Km and Vmax of the AnGSNOR for GSNO were calculated with Lineweaver-Burk plots. The effect of pH on GSNOR activity was measured in 100 mM acetate buffer (pH 4.0-5.5), 100 mM MES buffer (pH 5.5-6.5), 100 mM MOPS buffer (pH 6.5-7.5), 100 mM sodium phosphate buffer (pH 6.0-7.5), and 100 mM Tricine buffer (pH 7.5-9.0). The effect of temperature on GSNOR activity was determined at temperatures ranging from 20℃ to 60℃. The thermal stability of GSNOR was measured by activity measurements after incubating the enzyme at temperatures of 45℃, 50℃, or 55℃, for 4 min in sodium phosphate buffer (pH 6.0). For investigation of substrate specificity, Alb-SNO, cysteine-SNO, formaldehyde, acetaldehyde, propionaldehyde, methanol, ethanol, 2-propanol, and 1-butanol were tested as substrates.

Site-Directed Mutagenesis

Site-directed mutagenesis was carried out using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The amino acids Cys11, Cys48, Thr50, Cys100, Arg118, and Cys272 (A. nidulans GSNOR numbering) were all mutated to alanine (Ala). The targeted mutations and the integrity of the whole coding sequences were confirmed by sequencing analysis.

Product Analysis of GSNO Reaction

The products of the GSNO reduction were analyzed by HPLC (Agilent 1260, Agilent Technologies, USA) using a reverse-phase column (TSKgel ODS-100V, 4.6 × 15 cm; Tosoh Co., Ltd., Tokyo, Japan). The reaction mixtures contained 2 mM GSNO, 1 mM NADH, different concentrations of GSH (0, 1, 2, and 5 mM), and 3.2 μg of purified enzyme in a final volume of 1 ml. After incubation at 37℃ for 60 min, the reaction mixtures were filtered through a 0.22 μm pore filter (Merck Millipore, Darmstadt, Germany) and then applied to the column. Elution conditions were as follows: 0.5 ml/min flow rate at 25℃; solvent A, acetonitrile/formic acid (99.8/0.2 (v/v)); solvent B, water/formic acid (99.8/0.2 (v/v)); isocratic for 30 min with 10% solvent A and 90% solvent B. The eluent was monitored by determining the absorbance at 220 nm. GSNO and GSSG were estimated by relating the integrated chromatogram peak areas to those generated by similarly processed standard solutions of known solute concentrations. Ammonia was determined according to instructions and using the standard solution provided in the Sigma Ammonia Diagnostic Kit. Hydroxylamine was measured by the method described in Jensen et al. [15] with slight modification. In brief, 200 μl of reaction mixture was added to 200 μl of Na2HPO4 (100 mM, pH 4.4). Then 400 μl of 1% (v/v) 8-hydroxyquinoline (prepared in 50% ethanol) was added with mixing and finally 400 μl of 1 M Na2CO3 was added. The reaction mixture was heated at 95℃ for 5 min and cooled for 60 min, and the absorbance at 705 nm was determined. The hydroxylamine concentrations were calculated relative to 100 μM hydroxylamine standards.

Preparation of Albumin-SNO and Other Evaluations

Albumin-SNO was obtained by incubating 1 mg of bovine albumin in 1 mM NaNO2 containing 0.4 M HCl. Residual NaNO2 in the solution was removed using Micro Bio-Spin P6 columns (Bio-Rad Laboratories, Hercules, CA, USA). Albumin-SNO was confirmed using the Griess-Saville assay [37]. Protein concentration was determined with the Bicinchoninic acid (BCA) Protein Assay kit (Sangon Biotech, Shanghai, China) with bovine serum albumin as the standard. Amino acid sequence alignment was done with ClustalW and illustrated using ESPript.

 

Results

Sequence Analysis of A. nidulans GSNOR

In a previous study, Zhou et al. [37] identified a GSNOR-like gene (ANIA_07632) by BLAST research on the A. nidulans FGSC A4 genome database (Broad Institute, Cambridge, MA, USA) using Escherichia coli GSNOR as a query sequence. Here, we cloned and analyzed the full-length cDNA of the gene. Comparison of the genomic sequence with the cDNA sequence revealed that the gene is divided into six exons separated by five introns spanning about 2 kb, which agreed well with the information provided by Broad Institute. The gene encodes a protein consisting of 379 amino acids, which is highly homologous to those of GSNOR from E. coli (56.85%), Saccharomyces cerevisiae (66.67%), Arabidopsis thaliana (60.72%), and Homo sapiens (63.05%) [20,36] (Fig. 1A). The known GSNORs all contain two zinc atoms, one to activate substrates and the other to maintain protein structure [19,26]. As shown in the alignment, the amino acid residues that are predicted to coordinate with the catalytic zinc (Cys48, His70, Glu71, Cys177) and the structural zinc (Cys100, Cys103, Cys106, Cys114) in other GSNORs were also discovered in the fungal homolog. Moreover, the four residues essential for substrate binding (Thr50, Thr54, Asp59, Arg118) and the two residues (Cys11, Cys272) presumed to regulate GSNOR activity [36] are also well conserved in the fungal homolog. The high identity of amino acid sequences and the well-conserved characteristic residues suggest that the ANIA_07632 encoded protein belongs to the GSNOR family.

Fig. 1.Alignment and phylogenetic analysis of GSNOR amino acid sequences from A. nidulans and other eukaryote and prokaryote species. (A) Amino acid sequence alignment of GSNOR. The alignment of other GSNOR sequences from A. thaliana (NP_199207.1), E. coli (WP_024225747.1), H. sapiens (NP_000662.3), and S. cerevisiae (NP_010113.1). The amino acid residues involved in binding of the catalytic zinc atom, the structural zinc atom, and substrate are indicated by closed triangles, open triangles, and closed circles, respectively. The three amino acid residues presumed to have conserved functions in regulating GSNOR activity are indicated by open circles. (B) Phylogenetic relationships of GSNORs. The amino acid sequences of GSNORs from different species were aligned to construct a phylogenetic tree with MEGA 6.0, using the neighbor-joining method. Branch support values from 1,000 bootstrap replications are shown at each branch point. GenBank accession numbers are indicated in parenthesis.

We mapped the phylogenetic relationships between GSNOR homologs of prokaryotes and eukaryotes based on the deduced amino acid sequences (Fig. 1B). The phylogenetic tree shows that GSNOR proteins first diverged into eukaryotic and prokaryotic groups. The eukaryotic branch is further clearly separated into three major families – animals, plants, and fungi – each of which comprises proteins from extremely divergent organisms. Furthermore, the fungal family can be split into two subfamilies, yeasts and filamentous fungi. Sequences from numerous filamentous fungi, including A. nidulans, are members of the latter group. The well-conserved distribution of the GSNORs into the four branches according to the kingdom suggests that these proteins evolved vertically from a common ancestor.

Expression and Purification of Recombinant GSNOR

His-tagged AnGSNOR was produced as a recombinant soluble protein and purified using affinity chromatography. The purified AnGSNOR gave a single band with molecular mass of about 40 kDa on SDS-PAGE (Fig. 2A). The molecular size agreed well with the predicted molecular mass derived from the gene sequence, and was similar to other known GSNORs from E. coli (39.3 kDa), S. cerevisiae (41 kDa), and mouse (43 kDa) [15,19,20], which suggests that the GSNOR family may have evolved from a common ancestral protein. AnGSNOR migrated as a dimer in gel filtration, with an estimated molecular mass of 75 kDa (Fig. 2A). The homodimer form is also present in other GSNORs of known crystal structure [19], suggesting that the conserved dimer form may be necessary for enzyme activity.

Fig. 2.Purification of recombinant AnGSNOR and determination of its molecular mass. (A) SDS-PAGE analysis of the recombinant AnGSNOR. The purified enzyme was electrophoresed in a 12% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250. Lanes 1 and 2 are the protein molecular mass markers and the Ni-NTA column-purified protein, respectively. (B) Estimation of the molecular mass of AnGSNOR by Sephadex gel-filtration. Inset: correlation between the molecular mass and elution volume of protein standards (squares); AnGSNOR was eluted with an approximate molecular mass of 78 kDa (filled circle).

GSNOR Activity of AnGSNOR

A spectrophotometric kinetic assay that monitored absorbance changes at 340 nm due to GSNO-dependent NADH utilization [20] was used to measure the GSNOR activity of the purified AnGSNOR. As shown in Fig. 3A, when NADH and GSNO were mixed with AnGSNOR, the absorbance at 340 nm decreased, indicating an enzyme-dependent oxidation of NADH. Kinetic analysis gave an apparent Km for the substrate GSNO of 0.55 mM and an apparent kcat of 34,100 min-1. The apparent kcat was comparable to the values reported for the known GSNORs from human (kcat = 12,000 min-1), yeast (kcat = 52,600 min-1), and mouse (kcat = 5,600 min-1) [9,20], indicating that the recombinant protein was an efficient GSNOR.

Fig. 3.Biochemical properties of the recombinant AnGSNOR. (A) Consumption of GSNO by AnGSNOR. Inset: Lineweaver-Burk plot of the GSNO reduction data. The initial rate of the enzymatic reaction was determined at 400 μM NADH with various concentrations of GSNO (200-1,000 μM) in 100 mM sodium phosphate buffer, pH 6.0, at 37℃. Effect of pH (B), temperature (C), and thermal stability (D) on GSNOR activity. Optimum pH was determined at 37℃ in various buffers: sodium acetate (pH 4.0-5.5), MES (pH 5.5-6.5), MOPS (pH 6.5-7.5), sodium phosphate (pH 6.0-7.5), and Tricine (pH 7.5-9.0). The optimum temperature was determined in sodium phosphate buffer (pH 6.0) at various temperatures ranging from 20℃ to 60℃. The thermal stability of GSNOR was determined by measuring its activities under optimal conditions (100 mM sodium phosphate buffer, pH 6.0, 37℃) after exposing the enzyme to various high temperatures (45℃, 50℃, and 55℃, respectively) for 4 min. Error bars represent the SD of three independent experiments.

Because the reported GSNORs generate distinct products, multiple possible reaction mechanisms have been proposed. NH4+ and GSSG are the common products of the E. coli enzyme, and the yields depended on the concentration of exogenously added GSH [20]. In the case of rat and human GSNORs, the final measurable products are GSSG, NH4+, and hydroxylamine [15,29]. To define the reaction mechanism of fungal GSNOR, we investigated the product formation and distribution of the GSNO reduction catalyzed by AnGSNOR. As shown in Table 1, several measurable products, including GSSG, hydroxylamine, and NH4+, were detected. In the absence of externally added GSH, the GSSG yield was only about 28% of the input GSNO concentration. NH4+ also accumulated during GSNO processing. When an excess of GSH relative to GSNO substrate was added to the reaction mixture, abundant hydroxylamine and enhanced yields of GSSG were detected. However, the release of NH4+ was minimally affected by the added GSH. The appearance of hydroxylamine and the lack of influence of GSH addition on NH4+ production indicate that the reaction pathway of filamentous fungal GSNOR is closer to that of mammalian rather than that of E. coli enzymes.

Table 1.One milliliter of reaction mixture contained 3.2 μg of purified enzyme, 1 mM GSNO, 1 mM NADH, and different concentrations of GSH (0, 1, 2, and 5 mM). The mixture was incubated in 100 mM sodium phosphate buffer (pH 6.0) at 37℃ for 60 min. Results are the means ± SD of three independent experiments.

Substrate Specificity of AnGSNOR

GSNOR was originally identified as a GSH-dependent formaldehyde dehydrogenase (GSH-FDH), also known as a class III alcohol dehydrogenase (ADH3). This enzyme has long been proposed to detoxify endogenous and exogenous formaldehyde, due to its apparent ability to catalyze NAD+- and GSH-dependent oxidation of formaldehyde [15]. In order to determine whether A. nidulans GSNOR is capable of such a reaction, the activity of AnGSNOR towards various aldehydes and alcohols was assessed by monitoring the formation of NADH. As shown in Table 2, the catalytic efficiency of GSNO-dependent NADH oxidation was over 5-fold higher than that of formaldehyde-dependent NAD+ reduction, and about 20-fold higher than those of other aldehyde- and alcohol-dependent NAD+ reduction, suggesting that this dual catalytic enzyme preferentially functions as a GSNO reductase. The activity of AnGSNOR towards GSNO was also assessed by an NADPH oxidation assay, and the reaction rate reached about 55% of that observed for NADH (Table 2). Cysteine-SNO and albumin-SNO, representing other nitrosated peptides and proteins, were also tested as substrates for AnGSNOR activity, but in both cases the AnGSNOR activity was negligible. We thus concluded that the recombinant protein preferentially functions as a NADH-dependent GSNO-specific reductase.

Table 2.All reactions were carried out at 37℃ in 1 ml of 100 mM sodium phosphate buffer (pH 6.0) containing 1.6 μg of purified enzyme, 1 mM cofactor (NADH, NADPH, or NAD+), and 1 mM of each substrate. For reactions with formaldehyde, acetaldehyde, and propionaldehyde, the reaction mixtures also contained 1 mM GSH. The activity of GSNOR towards GSNO was taken as 100%. Results are the means ± SD of three independent experiments.

Enzymatic Properties (pH and Temperature Optima and Thermal Stability)

As shown in Fig. 3B, AnGSNOR activity was higher in sodium phosphate buffer than in MES or MOPS buffers. The optimal pH for enzyme activity was determined to be pH 6.0, with a sharp decline as pH decreased towards 4.0 or increased from 7.0 to 9.0. The effect of temperature on AnGSNOR activity was also investigated, and an optimum temperature of 37℃ was required to exhibit its maximal activity (Fig. 3C). The optimal pH and temperature were consistent with the typical growth conditions of this fungus, which might assure efficient protection by GSNOR against nitrosative stress. The thermal stability of the enzyme was also studied at three different temperatures at pH 6.0 (Fig. 3D). After a 4-min pre-incubation at 45℃, the enzyme still retained about 60% activity. However, pre-incubation at 50℃ for 4 min decreased the enzyme activity by nearly 90%, and incubation at 55℃ for 2 min inactivated the enzyme completely, indicating poor thermal stability of the enzyme.

Activities of AnGSNOR Mutants

As shown in Fig. 1A, the potential structural and catalytic zinc-coordinating residues and substrate-binding amino acids have been predicted based on the crystal structure of GSNORs from tomato, human, and Arabidopsis [19,26,36]. However, the real roles of the highly conserved residues have not been experimentally validated at all. Herein, site-directed mutagenesis was used to study the features of some conserved residues in the fungal GSNOR. Among all the alanine substitution mutants (Fig. 4), C48A of AnGSNOR displayed the lowest GSNOR activity, which suggested that this residue may have the conserved function of binding the catalytic zinc atom. Another mutant, C100A, was misfolded and completely localized to inclusion bodies, consistent with the prediction that Cys100 in other GSNORs is a structural zinc-coordinating residue to maintain structural stability of the enzyme. The mutants T50A and R118A of AnGSNOR, whose corresponding residues were predicted to involve binding of substrate, displayed 14% and 23% decreases in catalytic activity, respectively, compared with the wild-type AnGSNOR. GSNOR is a remarkably cysteine-rich protein with a mole percent cysteine of more than 3%, and about half of the cysteines are non-zinc-coordinating residues in the well-known organism GSNORs. Among them, two conserved cysteines – Cys11 and Cys272 – in human and Arabidopsis have been shown to be solvent-accessible based on the structure information, and had been predicted to have conserved functions in regulating GSNOR activity [36]. In AnGSNOR, the C11A mutant lost 46% catalytic activity, whereas the C272A mutant showed wild-type levels of catalytic activity, confirming the important role of Cys11 but not Cys272 in GSNOR activity regulation.

Fig. 4.Enzymatic activities of wild-type and mutant AnGSNORs. All the selected amino acid residues were substituted by alanine. Setting the enzymatic activity of wild-type GSNOR at 100%, the residual enzymatic activity of each of the mutants was determined. Data are the means ± SD of three independent experiments. I.E: insoluble expression.

 

Discussion

GSNORs are believed to regulate NO bioactivity, whether for signaling or defense, and have been isolated from bacteria, yeasts, and higher eukaryotes [5]. In this study, we characterized a recombinant GSNOR from a filamentous fungus in detail for the first time. The presence of GSNOR in a filamentous fungus demonstrates the ubiquitous distribution of the enzyme in organisms and fills the evolutionary gap between GSNOR in yeasts and that of higher eukaryotes.

A. nidulans GSNOR was much more specific towards NADH than NADPH in the GSNO reductase activity (Table 2), consistent with the cofactor preferences of human and yeast GSNORs [9]. NAD+/NADH and NADP+/NADPH redox couples are the major determinants of redox state in the cell. Under normal conditions, the cytoplasmic ratio of NAD+/NADH is about 700, whereas the ratio for NADP+/NADPH is much lower, which makes the cofactor pairs engage in distinct metabolic pathways [34]. In general, NAD+/NADH drives ATP production in the cytosol and mitochondria, whereas NADP+/NADPH governs lipid, amino acid, and nucleotide biosynthetic pathways and the defense against reactive oxygen species. Therefore, acting as a defense enzyme under cellular conditions, A. nidulans GSNOR may employ NADPH as the electron donor, although with relatively lower activity. There is another possible case in which GSNOR utilizes the preferential cofactor NADH, which can be immediately recycled by formaldehyde oxidation catalyzed by GSNOR functioning as an ADH3 with concomitant conversion of NAD+ to NADH [29]. The resulting NADH may not dissociate from the enzyme, but is directly used for GSNO reduction [8]. Thus, considering the usually low cytosolic free NADH/NAD+ ratio, the intracellular formaldehyde may trigger and promote GSNOR activity by enzyme-bound cofactor recycling.

Although microbes should combat with NO derived from animal immune systems, there is considerable evidence that endogenous NO in bacterial cells plays essential regulatory roles as a signaling molecule in a variety of biological processes [32]. Despite the absence of NOS-like sequences in eukaryotic microbial genomes, NO production has been proven in many fungi [1]. Therefore NO-dependent signaling pathways may also exist in fungi. The apparent kcat value of A. nidulans GSNOR is comparable to those of other reported GSNORs, which suggests that fungal GSNOR could feasibly regulate NO bioactivity for signaling purposes.

GSNOR, together with Trx systems, regulates the process of denitrosation of multiple proteins [4,27]. GSNOR reversibly nitrosates proteins via indirect transfer of NO between GSH and protein [10], because protein-SNOs are not the direct substrate of GSNOR (Table 2). In contrast, Trx/TrxR has a broad spectrum of denitrosated substrates, including various protein-SNOs, and also directly breaks down GSNO in vitro [31]. Additionally, under nitrosative stress conditions, TrxR was induced in Candida albicans [14] and deletion of Trx genes impaired the growth of Helicobacter pylori [7], Neisseria gonorrhoeae [7], and A. nidulans [37], indicating the important roles of the Trx system in NO resistance. However, in filamentous fungi, determination of whether Trx/TrxR really catalyzes intracellular GSNO and identification of the major physiological GSNO denitrosase among GSNOR, Trx/TrxR, and other candidate denitrosases (including xanthine oxidase, protein disulfide isomerase, superoxide dismutase, glutathione peroxidase, nitroreductase, and carbonyl reductase) await future investigation.