INTRODUCTION

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The wood-rotting basidiomycete fungus Phanerochaete chrysosporium degrades polymeric lignin (12, 21, 30) and various aromatic pollutants, including chlorinated phenols, dioxins, and polyaromatic hydrocarbons (9, 23, 26, 40). A wide variety of oxidized metabolic intermediates are generated during the degradation of lignin, lignin model compounds, and aromatic pollutants by this organism. These intermediates include substituted quinones, hydroquinones, benzaldehydes, benzoic acids, and ring-opened fragments (12, 15, 27, 30), which are metabolized further by intracellular processes (7, 42, 55). The extracellular peroxidases that are involved in the initial oxidative steps of lignin and pollutant degradation are well-characterized (15, 21, 27, 30); however, much less is known about the intracellular enzymes involved in the further degradation of monomeric intermediates, such as quinones, hydroquinones, and benzaldehydes. Recent work, including the elucidation of metabolic pathways for the degradation of several aromatic pollutants by P. chrysosporium (53, 54), has suggested that intracellular enzymes are involved in the reduction of quinones. Since benzoquinones are generated by the peroxidase-catalyzed oxidation of lignin and appear to be key intermediates in the degradation of aromatic compounds by P. chrysosporium (29, 42, 44, 51, 53, 54), we have been examining the reduction of benzoquinones by this organism.

The reduction of methoxylated, lignin-derived quinones by P. chrysosporium appears to be catalyzed by an intracellular quinone reductase (QR) or QRs (6, 7, 11, 13), and we have purified and partially characterized one intracellular, NAD(P)H-dependent QR from this organism (6, 7). The soluble protein is a 44-kDa dimer with two similar 22-kDa subunits and appears to contain two flavin mononucleotide (FMN) moieties per dimer. A variety of methoxylated quinones and other electron acceptors serve as substrates for this enzyme (6, 7). The stoichiometry of NADH oxidation to 2,6-dimethoxy-1,4-benzoquinone (DMBQ) reduction is 1:1, and the enzyme apparently catalyzes the reduction of quinones to hydroquinones via a ping-pong, steady-state, kinetic mechanism (6). NADH and NADPH are equally efficient as electron donors, and the enzyme is competitively inhibited, with respect to NADH, by both dicoumarol and Cibacron Blue (6). The latter properties are similar to properties reported for the mammalian QR DT-diaphorase (16, 17, 37).

Here, we describe the isolation and characterization of a cDNA clone encoding the P. chrysosporium QR, as well as studies on the regulation of QR synthesis.

	MATERIALS AND METHODS

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Culture conditions. Stock cultures of P. chrysosporium OGC101 (21), a derivative of strain BKM-F-1767 (unpublished data), were maintained on malt extract-yeast extract-Vogel medium slants as previously described (22). Stationary cultures were grown from conidial inocula either in 25 ml of medium in 250-ml flasks or in 50 ml of medium in 500-ml flasks for 2 days at 37°C (7, 42). The medium used has been described previously (31) and was supplemented with 2% glucose, 10 mM dimethyl succinate (pH 4.5), and either 12 mM (high-nitrogen [HN] medium) or 1.2 mM (low-nitrogen medium) ammonium tartrate as the nitrogen source. The 48-h stationary cultures were homogenized for 20 s in a Waring blender, and 100-ml portions of the homogenates were transferred to 250-ml flasks and grown at 28°C on a rotary shaker (150 rpm). After 48 h, inducers were added, and the cells were harvested at the times indicated below.

Isolation of the qr cDNA clone. A gt10 cDNA library was constructed from P. chrysosporium mRNA as described previously for a gt11 library (38). Cultures were grown in 1 liter of low-nitrogen medium as described above. Five-day-old cells were harvested by filtration, and RNA was isolated as described below and previously (8). Poly(A) RNA was isolated by passing the total RNA over oligo(dT)-cellulose (2). cDNA was synthesized as described previously (24, 38), and EcoRI adapter molecules were ligated to cDNA products (38, 43). Finally, cDNA products were ligated to GT10 arms, packaged in vitro, and amplified as described previously (38, 43). N-terminal sequencing of purified P. chrysosporium QR protein (7) was carried out by D. McMillen, Biotechnology Laboratory, Institute of Molecular Biology, University of Oregon. A fully degenerate 24-mer oligonucleotide, corresponding to the first eight N-terminal amino acids of QR (PKVAIIIY), was synthesized at the Oregon Regional Primate Research Center, Beaverton. The oligonucleotide was end labeled with [-³²P]dATP (Andotek) by using T4 polynucleotide kinase (New England Biolabs) and was used to screen plaque lifts of the library by standard methods (43). Positive plaques were purified, and the DNA was isolated with a Lambda Midi Kit (Qiagen) and digested with EcoRI to release the cDNA insert. The cDNA insert was subcloned into the plasmid vector Bluescript SKII⁺ (Stratagene) to generate pcQR1. Plasmid DNA was purified and sequenced in both directions with an Applied Biosystems Inc. model 373 stretch automated sequencer by using primer walking (47). A nucleotide sequence analysis was performed with MacVector 6.0 (Oxford, Molecular).

In vitro transcription and translation. pQRT1 was generated by isolating the 856-bp HincII-XbaI fragment from pcQR1 and ligating it into HincII-XbaI-restricted pBluescript SKII (Stratagene). Thus, in pQRT1 the most 5' ATG of the qr cDNA was 78 bp downstream of the start of the Bluescript T7 promoter. pQRT2 was constructed by isolating the 662-bp PstI-XbaI fragment from pcQR1 and ligating it into PstI-XbaI-digested pBluescript SKII (Fig. 1). Thus, in pQRT2 the downstream ATG of the qr cDNA was 130 bp downstream of the T7 promoter. The plasmid DNAs used for transcription-translation reactions were purified by using a Plasmid Midi Kit (Qiagen).

View larger version (62K): [in this window] [in a new window]   FIG. 1.   Nucleotide sequence of the P. chrysosporium qr cDNA. The amino acid sequence of the predicted translation product is shown below the nucleotide sequence. The qr coding region is flanked by a 59-bp 5' noncoding region and a 132-bp 3' noncoding region, excluding the poly(A) tail. The initiation codon is followed by an apparent 71-amino-acid leader sequence, which is underlined. The experimentally determined N-terminal amino acid sequence of the isolated QR protein is overlined. The two KEX2 cleavage sites in the leader sequence are enclosed in boxes. The dots indicate the potential glycosylation sites at residues 11 and 223.

Chemicals. The compounds used in induction experiments were obtained commercially and used directly. 2-Methoxybenzoquinone (MBQ) and DMBQ were prepared by silver oxide oxidation of the respective hydroquinones (25).

Enzyme extracts. Cells were harvested by vacuum filtration through Miracloth (Calbiochem), washed with ice-cold distilled water, and stored at 80°C. Frozen cells (1 g) were broken by grinding in a mortar and pestle with sand. Subsequently, extraction buffer (50 mM sodium phosphate [pH 7.0] containing 1 mM EDTA and 0.004% phenylmethylsulfonyl fluoride) was added with stirring. The homogenate was centrifuged at 17,300 × g for 20 min, and the resulting supernatant was centrifuged at 105,000 × g for 30 min.

Enzyme assays. The QR activity in the 105,000-×-g supernatant was measured by monitoring the oxidation of NADH at 340 nm. The standard reaction mixtures (1 ml) contained 50 mM sodium citrate (pH 6.0), 100 µM DMBQ, and enzyme (10 to 100 µl). Reactions were initiated by adding 200 µM NADH. Assays were carried out at room temperature by using a Shimadzu model UV260 spectrophotometer. Protein concentrations were determined by the bicinchoninic acid method (45) by using bovine serum albumin as the standard.

Immunoblot analysis. SDS-PAGE (Mini-PROTEAN; Bio-Rad) of the 105,000-×-g supernatant was performed by using a 12% polyacrylamide resolving gel as described previously (33). Electrophoretic transfers to nitrocellulose were carried out as described previously (49) by using a transfer apparatus (Bio-Rad). Rabbit polyclonal antibody was raised against purified QR as previously described (7). Nitrocellulose transfers were incubated with the rabbit antibody and then with goat anti-rabbit immunoglobulin G conjugated to alkaline phosphatase. Alkaline phosphatase activity was detected by the 5-bromo-4-chloro-3-indolyl phosphate nitroblue tetrazolium assay (5).

RNA preparation and Northern blot hybridization. Cells from induced and uninduced cultures were filtered through Miracloth, washed with cold distilled water, frozen in liquid N₂, and stored at 80°C. Total RNA was isolated by breaking the cells in 1.5-ml Sarstedt tubes containing 1 g of glass beads in the presence of 1 ml of TRI reagent (Molecular Research Center, Inc.) as previously described (8). After spectral quantitation, 20 µg of the RNA was denatured in 2.2 M formaldehyde containing 50% formamide for 15 min at 68°C and electrophoresed in a denaturing gel (0.22 M formaldehyde, 1% agarose) in buffer containing 0.22 M formaldehyde, as described previously (50). The RNA was transferred to a Magna NT membrane (Micron Separations, Inc.). pcQR1 and the P. chrysosporium glyceraldehyde-P-dehydrogenase (gpd) gene (35) were used as templates for randomly primed synthesis (18) of [-³²P]dCTP-labeled probes with a multiprime DNA labeling kit (Amersham). Northern blot hybridizations were performed as previously described (8).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and methods Results and discussion References

During degradation of polymeric lignin and aromatic pollutants by P. chrysosporium, substituted quinones are generated and reduced to hydroquinones, which undergo further metabolism (6, 7, 11, 13). Thus, the quinones and their corresponding hydroquinones are key intermediates in the degradative process (11, 29, 44, 51, 53, 54). Previously, we described purification and the reaction mechanism of an intracellular QR from P. chrysosporium, which is expressed during both primary and secondary metabolic growth (7). This enzyme reduces methoxyquinones and other substrates to their hydroquinones (6, 7). In order to better understand the mechanism and role of this enzyme in lignin and pollutant degradation, we cloned the qr cDNA from P. chrysosporium and studied the regulation of QR expression.

cDNA sequence. A degenerate oligonucleotide that was based on the experimentally determined N-terminal sequence of purified QR was used to probe a P. chrysosporium gt10 cDNA library. The sequence of the cDNA derived from a positive plaque and its predicted translation product are shown in Fig. 1. The cDNA consists of 1,007 nucleotides and a poly(A) tail. The 816-bp open reading frame encodes a 271-amino-acid protein and a TAG stop codon. The coding region is flanked by a 59-bp 5' noncoding region and a 132-bp noncoding region between the stop codon and the poly(A) sequence. The experimentally determined N-terminal sequence of QR, consisting of the first eight amino acids of the mature protein (PKVAIIIY), corresponds to the sequence of the predicted translation product of the cDNA from Pro-72 to Tyr-79 immediately following Met-71, indicating that this cDNA encodes the QR protein isolated (Fig. 1).

Translation start site. In order to determine experimentally whether translation of qr begins at Met-1 or Met-71, coupled transcription-translation of the cDNA was carried out by using ³⁵S-labeled Met, and the product was analyzed by SDS-PAGE and fluorography. When pQRT1 (in which the most 5' ATG encoding Met-1 is located 78 bp downstream of the T7 promoter) was used in the transcription-translation experiment, an abundant ³⁵S-labeled translation product was obtained (Fig. 2). This product had a molecular mass of ~27 kDa, which is within 5% of the calculated molecular mass of the larger predicted translation product. In contrast, when pQRT2 (in which the second ATG encoding Met-71 is located 130 bp downstream of the T7 promoter) was used in the transcription-translation experiment, no significant translation products were observed. Likewise, significant translation products were not observed in the control that lacked DNA (Fig. 2). These results indicate that translation can be initiated from the first ATG, although further work is needed to prove that translation cannot be initiated from the second ATG. The results also suggest that translation of the qr gene probably begins at the first ATG, which is located at nucleotide 60 in the cDNA sequence, and that QR is synthesized as a proenzyme with a 71-amino-acid leader sequence that is removed by enzymatic processing. The results further suggest that the putative KEX2 and/or dipeptidyl aminopeptidase sites observed in the sequence may be authentic cleavage sites. QR has been isolated as a cytosolic protein (7); however, very few cytosolic proteins contain leader sequences. There is a potential N-glycosylation site, conforming to the general rule Asn-X-Thr/Ser (32), beginning at amino acid 11 of the leader sequence (Fig. 1). If this site is glycosylated, then the proenzyme may be membrane bound via the leader sequence and the mature protein may be released to the cytosol or to an internal compartment during processing. There is also a putative N-glycosylation site at Asn-223. While no obvious membrane-spanning region is found in the leader sequence, there is a free cysteine at position 3 in the leader sequence, which may be a potential site for palmitoylation of this protein. Both G proteins (36) and Src proteins (41) are palmitoylated at a site near the N terminus.

View larger version (46K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE of the in vitro transcription-translation products obtained with the qr cDNA. Reactions and electrophoresis were performed as described in the text. Lane 1, control reaction mixture lacking DNA; lane 2, reaction mixture containing pQRT1, which included the upstream ATG of pcQR1; lane 3, reaction mixture containing pQRT2, which included only the downstream ATG of pcQR1. The prestained molecular mass markers used were carbonic anhydrase (34.8 kDa), soybean trypsin inhibitor (28.3 kDa), and lysozyme (20.4 kDa).

Induction of QR activity. Table 2 shows the effects on QR activity resulting from the addition of a wide variety of aromatic compounds to 3-day-old HN agitated cultures of P. chrysosporium. The most effective inducers of QR activity, vanillic acid and ferulic acid, are products of degradation of guaiacyl lignin by white rot fungi (28). The fact that QR activity was induced by ferulic and vanillic acids suggests that both of these compounds also are metabolized by P. chrysosporium during primary growth. Several quinones and hydroquinones also are effective inducers of QR activity. Veratric acid, which differs from vanillic acid in methylation at position 4, is a much weaker inducer than vanillic acid. Vanillin is a weaker inducer than vanillic acid, and vanillyl alcohol is not an effective inducer. All compounds that exhibit the 3-methoxy-4-hydroxy substitution pattern except vanillyl alcohol are effective inducers. These compounds may be good inducers in themselves, or they may be metabolized to methoxyquinones, which are effective inducers. The compounds listed in Table 2 which are not effective inducers of QR activity may not be converted readily to methoxyquinones. Most chlorophenols and nitroaromatic compounds are not effective inducers of QR activity; two exceptions are 4-chlorophenol and 2-nitrophenol (Table 2). 1,4-Benzoquinone is produced during metabolism of 4-nitrophenol by Moraxella sp., which also produces an inducible QR (46). Since the oxidized and reduced forms of various quinoid compounds are equally effective as inducers and since these forms probably undergo rapid interconversion, either one or both forms might be recognized by the regulatory system.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Time course for induction of QR activity. Vanillic acid (), MBQ (), or nothing () was added to 2-day-old HN cultures, as described in the text. Induced and uninduced cells were harvested at the times indicated, and the QR activity in the supernatants of crude extracts was assayed as described in the text.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Induction of QR activity with different concentrations of vanillic acid. Two-day-old HN cultures were induced with the indicated concentrations of vanillic acid. The cells were harvested after 16 h and broken, and QR activity was measured as described in the text.

Western blot analysis. An immunoblot analysis of QR from control and induced cultures was used to determine whether the increase in QR activity in induced cultures was due to an increase in the amount of QR protein or activation of preexisting enzyme. Figure 5 shows the concentration-dependent increase in the amount of QR protein that occurred 16 h after 0.5 or 2.0 mM vanillic acid was added. The results suggest that induction of QR occurs at the level of protein expression rather than via stabilization or activation of preformed enzyme.

View larger version (50K): [in this window] [in a new window]   FIG. 5.   Immunoblot analysis of QR from uninduced and vanillic acid-induced cells. Two-day-old HN cultures were not induced (lane 1) or were induced with 0.5 mM (lane 2) or 2.0 mM (lane 3) vanillic acid and then were harvested after 16 h, as described in the text. Portions (50 µg) of the supernatant proteins from crude cell extracts were loaded and electrophoresed on SDS-PAGE gels, transferred to nitrocellulose, and immunodetected, as described in the text.

Northern blot analysis. Northern blot analysis was used to determine whether the regulation of QR expression occurs at the level of RNA. Figure 6 shows a Northern blot of RNA extracted from uninduced HN cells and from HN cells induced with vanillic acid (2 mM) or MBQ (0.2 mM). A large increase in the amount of qr mRNA was observed 3 h after induction with either vanillic acid or MBQ. In contrast, the gpd transcript was not affected by these additions. This confirms the results shown in Fig. 5 and suggests that QR activity is regulated by both vanillic acid and MBQ or their metabolites at the level of mRNA, probably at the level of transcription. The fact that regulation of the qr mRNA by vanillic acid and MBQ corresponds to induction of QR activity indicates that the cloned cDNA encodes the QR protein isolated from P. chrysosporium. The mechanism of QR induction in P. chrysosporium may be similar to the mechanism of DT-diaphorase induction in mammalian cells, which is transcriptional (14, 48).

View larger version (28K): [in this window] [in a new window]   FIG. 6.   Northern blot of P. chrysosporium RNA probed with the qr cDNA and the gpd gene. Two-day-old HN cultures were not induced (lane 1) or were induced with vanillic acid (2 mM) (lane 2) or MBQ (0.2 mM) (lane 3), as described in the text. After 3 h, RNA were isolated from induced and uninduced cultures, electrophoresed, and transferred to membranes, as described in the text. The blots were probed with randomly primed 32P-labeled pcQR1, as described in the text. Then the blots were stripped and reprobed with 32P-labeled gpd.

View larger version (K): [in this window] [in a new window]   FIG. 7.   Clustal alignment of deduced protein sequences derived from the QR gene (this work), the brefeldin A resistance gene (bre) from S. pombe (52), and a gene (alt) encoding a minor allergen from Alternaria alternata (1). Identical portions of the sequences (bold) are enclosed in boxes; shading indicates sequence similarity.