INTRODUCTION

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In mammals, nitric oxide synthase (NOS), guanylate cyclase (GC), and (6R)-5,6,7,8-tetrahydrobiopterin (H₄B) biosynthetic enzymes are functionally linked (9, 15, 21). The well-accepted paradigm in mammals shows that nitric oxide (NO) generated by NOS binds to and activates GC, which converts GTP to cyclic guanosine-3',5'-monophosphate (cGMP), an intracellular mediator of a variety of cell functions. H₄B, a key cofactor in the NOS oxidation of arginine, is synthesized from GTP by a series of three enzymes, GTP cyclohydrolase 1,6-pyruvoyltetrahydrobiopterin synthase, and sepiapterin reductase (Fig. 1). These enzymes are also involved in a wide variety of physiological and pathophysiological processes in mammals (5, 23, 24). For example, both NOS and GTP cyclohydrolase 1, a rate-limiting enzyme for H₄B synthesis, are coinduced by the cytokines tumor necrosis factor alpha, interleukin-10, and interleukin-4 (23).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Biosynthetic pathway for H4B, and oxidation of products with I2/KI for fluorescence HPLC analysis.

The first bacterial NOS, designated NOS_NOC, was purified from a Nocardia species (strain NRRL 5646) and biochemically characterized in this laboratory (3, 4). NOS_NOC is active as a homodimeric, 110.6-kDa heme protein with a unique N-terminal amino acid sequence. NOS_NOC converts L-arginine to NO and L-citrulline via N^G-hydroxy-L-arginine, and its activity requires H₄B, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), NADPH, and Ca²⁺ as do mammalian NOSs. Like mammalian NOS, NOS_NOC is competitively inhibited by N^G-methyl-L-arginine and N^G-nitro-L-arginine. Although an NOS has been found in Physarum polycephalum (25), NOS_NOC is the only such enzyme system identified so far in bacteria.

cGMP has been detected in a variety of microorganisms, including Escherichia coli, Saccharomyces cerevisiae, Dictyostelium discoideum, and Bacillus licheniformis (7, 11-13, 17, 22). However, among prokaryotes, GC has been purified only from E. coli (12). Few investigations of possible biochemical roles of cGMP in microorganisms have been reported. cGMP appears to function as a cell cycle regulator and as an intracellular signaling factor in chemotactic responses (1, 5, 7, 8, 11, 14, 17, 22). H₄B and its biosynthetic enzymes have been found in variety of microorganisms (9, 24, 25). However, investigations of possible relationships of NOS, GC, and H₄B biosynthetic enzymes in bacteria have never been reported.

GTP plays a central role as a key biosynthetic intermediate for the synthesis of both cGMP and H₄B. GTP is of central importance in NOS-mediated processes. This compound is a precursor for both cGMP and, by a series of metabolic transformations, H₄B. The observation of a NOS enzyme system in prokaryotes raises new questions about the presence of supporting biochemical pathways that can both serve to produce essential cofactors for the NOS reaction and yield functionally active products that may play roles in cellular physiology, metabolism, and possibly pathogenicity. A paradigm for the centrality of GTP in NOS-related processes is summarized in Fig. 2. Here we present evidence for the presence of GC and H₄B biosynthetic enzymes and their products in Nocardia. We further provide results which suggest a relationship of these enzymes with NOS_NOC.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Paradigm showing involvement of GTP as a precursor for H4B and cGMP as they relate to NOS in Nocardia spp.

	MATERIALS AND METHODS

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Materials and reagents. (+)-Neopterin, biopterin, H₄B, phenylmethylsulfonyl fluoride (PMSF), benzamidine HCl, leupeptin, pepstatin A, creatine phosphate, CPK, akaline phosphatase (from bovine intestinal mucosa; EC 3.1.3.1.), dithioerythritol (DTE), dithiothreitol (DTT), GTP, cGMP, sodium nitroprusside, 3-isobutyl-1-methylxanthine, N^G-nitro-L-arginine, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), Dowex 1 × 2-400 (strong basic anion exchanger), and NADPH were purchased from Sigma Chemical Co. (St. Louis, Mo.). cGMP assay kits (product no. TRK-500) and L-sepiapterin were purchased from Amersham (Piscataway, N.J.) and Alexis Corporation (San Diego, Calif.), respectively.

Culture conditions. Nocardia sp. strain NRRL 5646 is maintained in the University of Iowa College of Pharmacy culture collection on sporulation agar (ATCC no. 5 medium). Cultures were grown by a two-stage fermentation protocol in 200 ml of sterile medium held in stainless steel-capped 1-liter DeLong culture flasks. The medium, which contained 1% (wt/vol) glucose, 1% (wt/vol) yeast extract, 0.5% (wt/vol) soybean flour, 0.5% (wt/vol) NaCl, and 0.5% (wt/vol) K₂HPO₄ in distilled water, was adjusted to pH 7.0 with 6 N HCl and then autoclaved at 121°C for 20 min. Cultures were incubated with shaking at 250 rpm at 28°C on a Gyrotory shaker (Inova 5000; New Brunswick Scientific, Edison, N.J.). A 10% inoculum derived from 72-h-old stage I culture was used to initiate stage II cultures. Stage II cultures were harvested after 48 h by centrifugation at 10,000 × g (Sorvall RC-5; DuPont Co., Newton, Conn.) for 10 min, and the pellets were stored at 20°C.

Protein assay. Protein concentrations were measured by the modified Bradford microassay (Pierce, Rockford, Ill.), using bovine serum albumin as the standard (2).

GC activity. (i) In cell extracts. GC activity was determined by a modification of the method of Stone and Marletta (21). For preparation of cell extracts, 9 g (wet weight) of cell pellet was suspended in 15 ml of 50 mM Tris buffer (pH 7.0) containing 1 mM PMSF, 50 mM NaCl, 5 mM DTT, 1 mM benzamidine, 1 µg of leupeptin per ml, and 1 µg of pepstatin per ml. Cell disruption was achieved by sonication at full power over ice for 5 min, using 1-min intervals between 1-min sonication pulses, with a Sonifier Cell Disrupter 350 (Branson Sonic Power Co., Danbury, Conn.). Cell debris was removed by centrifugation at 100,000 × g for 60 min at 4°C, and the supernatant was used directly for GC assays. The standard, freshly prepared incubation buffer (210 µl) contained 50 mM Tris-HCl (pH 7.0), 1 mM 3-isobutyl-1-methylxanthine, 1 mM DTT, 0.1 mM GTP, 1 mM MnCl₂, 15 mM creatine phosphate, and 30 µg of CPK. Reactions were initiated by adding 90 µl of cell extract, and incubations were continued for 10 min at 37°C before being terminated by the addition of 1.7 ml of cold 95% ethanol. The resulting mixtures were centrifuged for 10 min in a Microfuge. Supernatants were dried in a SpeedVac Concentrator RVT 1000 (Savant, Holbrook, N.Y.), and the residues were dissolved in 100 µl of 50 mM Tris buffer (pH 7.4) containing 1 mM EDTA. cGMP concentrations in reconstituted samples were measured by radioimmunoassay (cGMP assay kit TRK 500; Amersham) (21).

(ii) In culture medium. Concentrations of cGMP in culture medium samples were determined by a modification of the method of Shibuya et al. (18-20). Stage II Nocardia cultures were harvested after 48 h of incubation by centrifugation at 10,000 × g for 10 min. Perchloric acid (1 ml of 60%) was added to 20 ml of culture supernatants; the mixtures were incubated for 5 min at 4°C and then centrifuged at 10,000 × g for 10 min. Activated charcoal (0.5 ml of 10% [wt/vol] in water; Merck, Rahway, N.J.) was added; after standing for 5 min at 4°C, mixtures were centrifuged at 10,000 × g for 30 min. Precipitates were mixed with Celite (1 ml of 50% [wt/vol] in water), and the resulting suspensions were loaded onto Celite columns (0.6 by 1.5 cm). Columns were washed sequentially with 3 ml of water and then 3 ml of 0.3 N NH₄OH in 50% aqueous ethanol. Ammonia-ethanol eluates were diluted with 3 ml of water and loaded onto Dowex 1 × 2-400 (0.6 by 1.5 cm; Sigma) columns that were each eluted with 4 ml of 0.5 N HCOOH and then 4 ml of 4 N HCOOH. The 4 N HCOOH eluates were freeze-dried; the residues were dissolved in 1-ml volumes of 20 mM ammonium formate (pH 6.5) and loaded onto alumina (Fisher Scientific Co. A-540) columns (0.6 by 1.5 cm) that were eluted with 3.5 ml of 20 mM ammonium formate. The eluates were lyophilized, the residues were dissolved in 200 µl of 50 mM Tris buffer (pH 7.4) containing 1 mM EDTA, and 100-µl volumes were analyzed for cGMP as described earlier. Standard curves were run with each set of unknown assay samples by assaying 20-ml volumes of autoclaved culture medium containing cGMP (0, 1.25, 2.5, 5.0, and 10 pmol). All assays were conducted in duplicate.

HPLC. Samples were analyzed with a Shimadzu HPLC (high-performance liquid chromatograph; LC 10 AD) equipped with a fluorescence detector (RX 10AXL) set with the excitation wavelength at 350 nm and the emission wavelength at 450 nm. Analytical HPLC separations were achieved over an octadecyl silica reverse-phase column (5 µm, 4.6 by 25 mm; Alltech, Deerfield, Ill.). Samples were resolved using an isocratic solvent system of 10% methanol in H₂O at an elution rate of 1 ml/min. Under these conditions, retention volumes (R_v) for authentic standards of neopterin and biopterin were 3.7 and 5.9 ml, respectively. Preparative HPLC separations were carried out over an octadecyl silica column (5 µm, 22.5 by 250 mm; Alltech) using the same solvent system at an elution rate of 3 ml/min. The R_v for neopterin in this system was 21.69 ml.

HPLC-MS. HPLC-mass spectra were obtained with an HP 1100 LC/MSD (Hewlett-Packard, Palo Alto, Calif.). HPLC separations were carried out as above. The mass range for detection was set between 200 m/z and 300 m/z. For direct mass spectrometry (MS) measurements, samples were introduced by a flow injection method using 50% CH₃CN in 50% H₂O with 0.05% trifluoroacetic acid and a detection range from 0 to 1,500 m/z.

H₄B biosynthetic enzyme activities. GTP cyclohydrolase 1 and sepiapterin reductase activities were determined by a modification of the method of Werner-Felmayer et al. (25).

GTP cyclohydrolase 1. Nocardia cells (9 g [wet weight]) were suspended in 10 ml of 50 mM Tris buffer (pH 8.0) containing 1 mM PMSF and 5 mM dithioerythritol (DTE) and disrupted by sonication at full power for 5 min over ice. Cell debris was removed by centrifugation at 100,000 × g for 60 min. Cell extract (3.84 mg/ml, 3 ml) was added to a Sephadex G-25 column (1.5 by 40 cm) which was eluted with 100 mM Tris buffer (pH 8.0) containing 5 mM EDTA, 1 mM PMSF, and 5 mM DTE while being monitored at 280 nm. A 3-ml sample of the initial protein eluate (3.4 mg of protein per ml) was mixed with 2 mM GTP and incubated in the dark at 37°C for 90 min. Samples of 125 µl of 1% I₂ and 2% KI in 1 N HCl were added to the reaction mixture, which was incubated for 1 h at room temperature and centrifuged in a Microfuge for 5 min.

Sepiapterin reductase. For this assay, 3 ml of the 100,000 × g cell extract was passed through a Sephadex G-25 column (1.5 by 40 cm) eluted with 100 mM phosphate buffer (pH 6.8). A 1-ml sample of the initial protein eluate (3.3 mg/ml) was incubated with 50 µM sepiapterin and 100 µM NADPH in the dark at 37°C for 30 min. This reaction mixture was subjected to the same sequence of I₂ oxidation, ion-exchange column, and HPLC analysis for biopterin as described above. Specific activity was expressed as picomoles of biopterin formed per hour per milligram of protein.

Conversion of GTP to H₄B. For this analysis, 3 ml of the 100,000 × g cell extract was passed through a 1.5- by 40-cm Sephadex G-25 column eluted with 100 mM Tris buffer (pH 7.4) containing 12 mM MgCl₂, 1 mM PMSF, and 5 mM DTE. A 3-ml sample of the initial protein eluate (3.2 mg/ml protein) containing 2 mM GTP and 2 mM NADPH was incubated in the dark at 37°C for 90 min. The resulting reaction mixture was processed as described above and analyzed by fluorescence HPLC for both neopterin and biopterin (Fig. 4). Specific activities for biosynthesis of neopterin and biopterin were expressed as picomoles of neopterin or biopterin formed per hour per milligram of protein, respectively.

Isolation of neopterin from a preparative-scale GTP cyclohydrolase 1 reaction. Wet cells (70 g) were disrupted in 50 ml of 50 mM Tris buffer (pH 8.0) containing 1 mM PMSF and 5 mM DTE by sonication at full power for 5 min over ice. After centrifugation at 100,000 × g for 60 min, the cell extract (50 ml) was loaded onto a Sephadex G-25 column (3 by 60 cm), and the column was eluted with 100 mM Tris buffer (pH 8.0) containing 5 mM EDTA, 1 mM PMSF, and 5 mM DTE. GTP (2 mM) was added to a 40-ml sample of the initial protein eluate, and the reaction mixture was incubated in the dark at 37°C for 90 min. After samples of 1% I₂ and 2% KI in 1 N HCl (5 ml) were added, the mixture was incubated for 1 h at room temperature and centrifuged at 10,000 × g for 10 min. The supernatant was titrated with 0.1 N ascorbic acid until I₂ color disappeared, neutralized with 1 N NaOH, treated with 2,000 U of alkaline phosphatase, and incubated at 37°C for 1 h in the dark. The reaction mixture was loaded onto a Dowex 50 (10 ml, H⁺ form; Sigma) column and then washed with 100 ml of H₂O and 50 ml of 1 N NH₄OH. The fraction eluted with 1 N NH₄OH was neutralized with glacial acetic acid, and the solution was concentrated to 10 ml. The solution (1 ml) was injected into an HPLC equipped with a semipreparative column, and fractions containing neopterin (by fluorescence detection) were collected. The isolated fraction was identical by thin-layer chromatography (reverse phase, methanol:H₂O = 2:8) to authentic neopterin and analyzed by direct-injection electron spray MS (negative ion mode).

	RESULTS

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Nocardia GC activity and extracellular cGMP. Cell extracts of Nocardia species prepared by sonication typically contained 4.1 mg of protein/ml. To measure cGMP activity, incubation mixtures contained 3-isobutyl-1-methyl-xanthine as an inhibitor of possible phosphodiesterase activity to ensure that cGMP formed was not degraded as it was being formed. Likewise, CPK and creatine phosphate were present as components of a GTP regeneration system to ensure that levels of the substrate, GTP, were adequate. Assays were routinely conducted for 10 min because cell extract incubations showed reproducible, measurable, and linear and time-dependent increases in cGMP over a 20-min reaction time. Based on three sets of duplicate experiments, GC specific activity was measured at 9.36 ± 0.46 pmol h¹ mg of protein¹ cGMP from GTP.

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View larger version (14K): [in this window] [in a new window]   FIG. 3.   Effects of additions to Nocardia cultures of arginine (Arg), GTP, H4B, sodium nitroprusside (SNP), nitro-L-arginine, and ODQ on culture medium cGMP concentrations.

Determination of activities of pteridine biosynthetic enzymes. Assays for GTP cyclohydrolase 1, the first step in H₄B biosynthesis, were carried out in 100,000 × g cell extracts that contained 5 mM EDTA to trap MgCl₂ as a means of preventing 6-pyruvoyltetrahydrobiopterin synthase from converting 7,8-dihydroneopterin triphosphate to 6-pyruvoyltetrahydrobiopterine (Fig. 1). Oxidation with I₂/KI and hydrolysis with alkaliine phosphatase converted 7,8-dihydroneopterin triphosphate to neopterin, suitable for fluorescence HPLC and MS analysis. HPLC chromatograms of GTP cyclohydrolase 1 assays typically showed two fluorescent peaks at R_v 1.9 and 3.7 ml. The major peak at 3.7 ml was identified as neopterin by comparison with an authentic standard. For further confirmation of neopterin as a product of the enzyme incubation mixture, analysis was also conducted using HPLC-electron spray MS in the positive ion mode. Peaks were observed at m/z (percent relative intensity) 235.9 (M⁺  H₂O + 1, 30%), 253.8 (M⁺ + 1, 60%), 276.0 (M⁺ + Na⁺, 7,289 (162 + I, 100%). In addition, neopterin isolated by preparative HPLC from a preparative-scale 40-ml reaction was identified by comparison with authentic neopterin by thin-layer chromatography and direct-inlet electron spray mass spectrometry operating in the negative ion mode. The results showed m/z (percent relative intensity) 378.9 (M⁺ + I  1, 100%), 514.9 (M⁺ + I + CF₃COOH  1, 75%), 651.0 (M⁺ + I + 2 × CF₃COOH  1, 28%), 786.8 (M⁺ + I + 3 × CF₃COOH  1, 12%). Based on HPLC analysis, GTP cyclohydrolase 1 activity was measured as 77.5 pmol of neopterin h¹ mg of protein¹.

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View larger version (14K): [in this window] [in a new window]   FIG. 4.   HPLC chromatogram showing the elution of neopterin and biopterin obtained by conversion of GTP by Nocardia cell extract.

	DISCUSSION

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A unique NOS enzyme system has been isolated from Nocardia sp. strain NRRL 5646 and characterized (3, 4). As in mammals, the enzyme catalyzes the NADPH-, H₄B-, FAD-, FMN-, O₂-, and Ca⁺²-dependent oxidation of arginine to citrulline and NO. In mammals, GTP is a centrally important biosynthetic precursor for cGMP and for H₄B. These two GTP-derived substances are important in providing a reducing substance (H₄B) for the NOS reaction and a product whose biosynthesis is greatly enhanced as a consequence of NO binding to the enzyme GC. Interestingly, enzymes involved in the biosynthesis of cGMP and H₄B are coinduced by cytokines in mammals (23). Since NOS_NOC was first discovered in the bacterium Nocardia, it was logical to examine the possibility that an NO-stimulated GC existed in this organism and whether the enzymatic capacity for H₄B biosynthesis could be demonstrated. Activities for GC and H₄B biosynthesis enzymes were found in the soluble, 100,000 × g supernatant fractions of cell preparations.

When NO is produced by NOS, it reacts with GC and activates the enzyme 20- to 100-fold. In mammals, cGMP has been well established as an intracellular mediator, through NOS, of a variety of cell functions. However, few studies have focused on roles of cGMP in bacteria. In E. coli, cGMP is secreted into the medium and its concentrations increase parallel with growth (18, 19). Cook et al. reported transient increases of intracellular concentrations of cGMP at different stages of E. coli growth and suggested that cGMP functioned as a cell cycle regulator (6). Interestingly, chemoattractants transiently activated GC in the mold D. discoideum (11).

GC activity was clearly present in 100,000 × g cell extracts of Nocardia sp. strain NRRL 5646 measured at 9.56 pmol of cGMP h¹ mg of protein¹. The activity measured for Nocardia sp. was less than that observed in cell extracts of E. coli (13), which showed specific activities of 24 to 108 pmol of cGMP h¹ mg of protein¹. Little has been reported on the properties of GC from prokaryotes. Although a single GC was identified in 1975 in E. coli, no structural information concerning amino acid or gene sequences for bacterial GCs have been reported (12).

The measured concentration of cGMP in control culture medium was 45 pmol of cGMP liter¹. When GTP, a biosynthetic precursor for both cGMP and H₄B, was added to culture medium, the concentration of cGMP increased to 180 pmol per liter of culture medium. While addition of L-arginine and H₄B separately caused slight increases of cGMP, to 50 and 80 pmol per liter of medium, respectively, L-arginine and H₄B together dramatically increased concentrations of cGMP, to 315 pmol liter¹. These results are consistent with our finding that omission of H₄B results in complete loss of NOS_NOC activity (3). The unexpectedly moderate increase of cGMP to 80 pmol per liter of medium by sodium nitroprusside, a chemical NO donor, may be due to impermeability of NO through Nocardia cell membranes or to the chemical or enzymatic instability of NO in the incubation medium. Results with nitroprusside also support the suggestion that cGMP increases are due to a Nocardia NOS. Furthermore, the fact that the NOS_NOC inhibitor N^G-nitro-L-arginine reduced cGMP levels to zero also indicated that NO generation by NOS_NOC is required for activation of GC.

The cell extract of Nocardia contained specific activities for GTP cyclohydrolase 1 and sepiapterin reductase of 77.5 pmol of neopterin and 45.8 pmol of biopterin h¹ mg of protein¹, respectively. The products from the two enzyme assays were also characterized by HPLC-MS. The total activity assay for H₄B biosynthesis showed two products, neopterin and biopterin. These results suggest that GTP was converted to H₄B through 7,8-dihydroneopterin triphosphate and then L-sepiapterin.

In conclusion, GC and H₄B biosynthetic enzyme activities are present in cell extracts of Nocardia sp. strain NRRL 5646. The changes of concentrations of cGMP in culture media by additions of GTP, L-arginine, H₄B, sodium nitroprusside, N^G-nitro-L-arginine, and ODQ indicate that GC is activated by NO generated by NOS_NOC, which requires H₄B as a cofactor. The results of this study support the proposed paradigm (Fig. 2) for a Nocardia NOS system that utilizes metabolic products of GTP metabolism to enhance NOS oxidation of arginine and to enhance GC to form cGMP. This work raises new and interesting questions regarding the nature of Nocardia GC and the possible roles of cGMP in Nocardia spp.