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Penicillium roqueforti is a fungal species traditionally used in the ripening of French Roquefort cheese. Since the discovery of PR toxin by Wei et al. (20), several other secondary metabolites related to PR toxin by P. roqueforti, such as eremofortins A, B, C, D, and E, have been isolated and characterized (2, 9-11). Recently, we found that another secondary metabolite (PR acid) is produced by P. roqueforti (6). Among these metabolites, only the PR toxin is lethal to rats, mice, and cats (7, 17). PR toxin inhibits RNA and protein synthesis (13, 14); the activities of the DNA polymerases , , and  (8, 18); and mitochondrial respiration and oxidative phosphorylation in animal cells (18, 22). It also alters the genetic activities of Saccharomyces cerevisiae and Neurospora crassa (19). The disappearance of PR toxin has been associated with an increase in PR acid (6). Moreover, the structures of the PR toxin (C₁₇H₂₀O₆) and PR acid (C₁₇H₂₀O₇) are closely related. Both compounds differ by only an aldehyde (PR toxin) and a carboxyl group (PR acid) at the C-12 position (6, 16). In previous studies, we established that eremofortin C is the precursor of PR toxin (3) and that eremofortin C is transformed into PR toxin by eremofortin C oxidase (21). Using these studies as models, we discovered that an enzyme in the culture medium of the fungus was responsible for the transformation of PR toxin into PR acid. Therefore, this study was designed to isolate, purify, and characterize this enzyme. Our results may elucidate the metabolic pathway of PR toxin in the culture medium of P. roqueforti.

P. roqueforti ATCC 48936 was maintained on potato dextrose agar slants at 4°C. To obtain high yields of PR toxin and other secondary metabolites, we grew the fungus in YESC medium, which contains 1% yeast extract, 7.5% sucrose, and 20% corn extract (4). Liquid medium (150 ml) was incubated in a Roux bottle (600 ml) to a final concentration of 5 × 10⁴ spores/ml and then incubated at 24°C as a stationary culture. PR toxin and PR acid were isolated from the culture medium and further purified by a previously described method (6, 20).

The amounts of PR toxin and PR acid were quantitatively analyzed by high-performance liquid chromatography (HPLC). A model 510 liquid chromatograph equipped with a model 486 tunable absorbance detector set at 254 nm was used with a model 740 data module (all from Waters). Separation was achieved with a Cosmosil 10 SL column (25 cm by 4.6 mm) for PR toxin and a Cosmosil 10 C₁₈ column (25 cm by 4.6 mm) for PR acid. PR toxin was measured with chloroform as the solvent at a flow rate of 2 ml/min, as described by Moreau et al. (12). PR acid was assayed with methanol-H₂O (2:3) containing 20 mM citrate-phosphate buffer (pH 3.0) as the mobile phase at a flow rate of 1.5 ml/min (6). The retention times of PR toxin (2 min) and PR acid (8.8 min) were verified with their respective standards. The amounts of these two compounds in the sample were determined by measuring the peak area of each compound in the chromatogram against the peak of a known standard.

The activity of PR oxidase was determined by HPLC analysis of the amount of PR toxin that converted to PR acid with a suitable amount of the enzyme. The assay mixture, in a total volume of 10 ml, contained 6.2 µmol of PR toxin (dissolved in 45 µl of methanol), 0.5 ml of the enzyme solution, and 20 mM citrate-phosphate buffer (pH 5.0). The reaction proceeded at 30°C for 10 min and then terminated after the addition of 5 ml of chloroform-acetic acid (6:1). After thorough mixing, the organic layer was collected. An aliquot of 20 µl was injected into an HPLC column for the measurement of the amount of PR acid that was converted from PR toxin by the enzyme. One unit of the enzyme activity was defined as the amount of enzyme that catalyzed the transformation of 1 µmol of PR toxin to PR acid per min under the assay conditions.

The culture medium of P. roqueforti was collected on day 22, when the PR oxidase activity reached maximum. Spores were removed by centrifugation at 9,000 × g for 20 min. The supernatant (7 liters) was concentrated by use of a polyfiber ultrafiltration system with a type SIY 10 membrane cartridge having a 10,000-molecular-weight cutoff (Amicon, Inc.). The concentrate was then slowly mixed with ammonium sulfate to 80% by constant stirring at 4°C, and the final precipitate was dissolved in 20 mM citrate-phosphate buffer (pH 5.0). The resultant enzyme solution was dialyzed against the same buffer. The dialysate was put onto a Q-Sepharose column (5.5 by 20 cm) preequilibrated with 20 mM citrate-phosphate buffer (pH 5.0). After being washed with 2 bed volumes of the column buffer, the column was eluted with a stepwise gradient of 50, 100, 250, and 500 mM NaCl in the same buffer (300 ml each). The fractions containing the enzyme activity were pooled and concentrated with a membrane filter apparatus with an XM 50 membrane (Amicon, Inc.). The enzyme solution was chromatographed on a Phenyl-TOYOPEARL 650 M column (1.6 by 30 cm) equilibrated with 20 mM citrate-phosphate buffer (pH 5.0) containing 2 M (NH₄)₂SO₄. The enzyme was eluted by lowering the ionic strength of the (NH₄)₂SO₄. The active fractions that eluted with 0 M (NH₂)₂SO₄ were combined and concentrated as in the previous step. The concentrate was further purified by analytical gel filtration on an HPLC by using a Bio-Sil SEC-250 column (0.78 by 30 cm; Bio-Rad, Richmond, Calif.). Elution was performed with 20 mM citrate-phosphate buffer (pH 5.0) containing 0.25 M NaCl.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 7.5% polyacrylamide gel and isoelectric focusing were performed as described previously (21). For examination of the glycoprotein nature of PR oxidase, the purified enzyme was subjected to periodic acid schiff staining according to the method of Thotakura and Bahl (15). To estimate the sugar content of PR oxidase, the purified enzyme was deglycosylated as described previously (21). The enzyme was incubated in a solution containing 0.2 U of endoglycosidase (Endo F; Sigma Chemical Co., St. Louis, Mo.), 0.1% SDS, 1% Triton X-100, 50 mM EDTA, 1% 2-mercaptoethanol, and 50 mM sodium phosphate (pH 5.0). The reaction was conducted at 37°C for 5 h. These conditions were chosen to demonstrate complete deglycosylation. The decrease in molecular mass compared to that of the native enzyme was considered the mass of the sugar residue of PR oxidase. Protein concentrations were determined as described by Bradford (1) with a Bio-Rad protein assay kit.

In our previous study (6), we observed that PR toxin appeared earlier than PR acid and that with time the amount of PR toxin decreased while that of PR toxin concomitantly increased. When PR toxin was incubated in the buffer solution containing the culture medium that had been grown with P. roqueforti, PR acid appeared and the amount of PR toxin decreased. These results suggest that the culture medium contains the enzyme system that is responsible for the transformation of PR toxin into PR acid. A similar finding was made with the crude extract of the mycelium of the fungus. When we examined the activity of the enzyme during the growth period of the fungus, we found that maximum activity occurred on day 22 of the culture.

When the enzyme activity reached maximum, the culture medium was harvested. The purification of PR oxidase is shown in Table 1. The crude enzyme, after concentration by ultrafiltration, was subjected to a series of purification procedures, including ammonium sulfate fractionation, ion-exchange chromatography on a Q-Sepharose column, hydrophobic chromatography on a Phenyl-TOYOPEARL 650 M column, and gel chromatography on an HPLC Bio-Sil SEC-250 column. The PR oxidase was purified 6.5-fold, giving a 10.2% yield. The specific activity of the final preparation was 201 U/mg. The purified PR oxidase showed a single band by SDS-PAGE (Fig. 1).

View larger version (51K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of the purified PR oxidase. Lane 1, molecular mass markers; lane 2, purified PR oxidase. The proteins were stained with Coomassie brilliant blue.

There were three major protein peaks when the enzyme was purified on an HPLC Bio-Sil SEC-250 column, but only one showed oxidative activity. The native molecular mass of this peak was ca. 90,000 Da. SDS-PAGE revealed that the molecular mass of the PR oxidase was approximately 88,000 Da, which indicates that the native enzyme is a monomer. The enzyme was shown to be a glycoprotein by periodic acid schiff staining. By use of a commercial endoglycosidase, we were able to remove the sugar residues from PR oxidase. The truncated enzyme molecule had a molecular mass of 67,000 Da, which indicated that the mass of the sugar residues was about 21,000 Da and accounted for 23.9% of the molecular mass of the native enzyme molecule. When analyzed by isoelectrofocusing in the ampholine polyacrylamide gel, PR oxidase exhibited a single band. The pI of PR oxidase was estimated to be 4.5, which indicates that the enzyme is an acidic protein.

As shown in Fig. 2A, the enzyme showed maximum activity at pH 4.0. The activity of the enzyme was estimated at various temperatures between 30 and 80°C. The results showed that the optimum temperature for the oxidation of PR toxin was 50°C (Fig. 2B). The enzyme was incubated at various temperatures for 20 min, and the remaining enzyme activity was measured under standard conditions. The enzyme was stable below 60°C. Thirty-eight and 23% of initial activity remained after incubation at 70 and 80°C, respectively. The enzyme activity was observed in the pH range of 3.0 to 9.0 upon incubation at 30°C for 30 min. The enzyme activity was stable between pHs 4 and 6. Approximately 50% of the activity was retained at pHs 3.0 and 7.0. Only a very small fraction of the activity remained at pH 9.0. 

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Effects of pH (A) and temperature (B) on the activity of PR oxidase from P. roqueforti. (A) Enzyme activity was assayed under the standard assay conditions except that the following buffers (20 mM) were used: citrate-phosphate (-), sodium phosphate (-), and Tris-HCl (-). (B) Assays were performed at various temperatures under the standard enzyme assay conditions. Relative activity is expressed as a percentage of the maximum activity.

Since the enzyme catalyzed the oxidation of the -CHO group of PR toxin to the -COOH group of PR acid (Fig. 3), the enzyme must be either an aldehyde dehydrogenase or an oxidase. In this study, we found that the enzyme does not require NAD⁺ or NADP⁺ as a cofactor to catalyze the transformation. By using the coupling assay in the presence of peroxidase and 4-aminoantipyrine plus phenol, we were able to determine the oxidase activity of the enzyme. The oxidase activity was found to be dose dependent and correlated very well with the transforming activity of the enzyme. Thus, the transforming enzyme is probably an oxidase.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Transformation of PR toxin into PR acid by the PR oxidase.

PR toxin was the first and only toxic secondary metabolite of P. roqueforti (7, 17). In the past few years, we have come across several secondary metabolites related to PR toxin and observed that they are produced sequentially in the culture medium of the fungus (5, 6). The compounds were isolated, purified, and identified as PR-imine (C₁₇H₂₁O₅N), PR acid (C₁₇H₂₀O₇), and PR-amide (C₁₇H₂₁O₆N), respectively. Moreover, we found that eremofortin C is transformed to PR toxin by EC oxidase (21). As part of our effort to understand the degradative pathway of the toxin, we discovered and purified the oxidizing enzyme that catalyzes the conversion of PR toxin to PR acid. In addition, eremofortin C was added to the assay system to see whether PR oxidase could oxidize eremofortin C to PR acid. The negative results indicated that PR oxidase is specific for PR toxin.