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Mn2+ is dispensable for the production of active MnP2 by Pleurotus ostreatus
BBRC Biochemical and Biophysical Research Communications 327 (2005) 871–876 www.elsevier.com/locate/ybbrc
Mn2+ is dispensable for the production of active MnP2 by Pleurotus ostreatus Hisatoshi Kamitsujia, Yoichi Hondaa,*, Takashi Watanabea, Masaaki Kuwaharab a
Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan Institute of Wood Technology, Akita Prefectural University, Kaieizaka, Noshiro, Akita 016-0876, Japan
b
Received 30 November 2004 Available online 24 December 2004
Abstract The regulation mechanism for expression of versatile peroxidase MnP2 by the basidiomycete fungus Pleurotus ostreatus was examined using chemically defined synthetic media. Expression of MnP2 was down-regulated at the transcription level by nutrient nitrogen, e.g., NHþ 4 , arginine or urea. As is often the case with other fungal manganese peroxidases, active MnP2 was not detected when Mn2+ was omitted from the culture, while mnp2 transcription was barely affected by Mn2+. However, Mn2+ can be substituted by an MnP2 substrate, Poly R-478, since active MnP2 was detected extracellularly when the compound was added to the culture without Mn2+. Enzyme stability assays with the purified MnP2 indicated an indispensable requirement for a substrate that can be used to complete the catalytic cycle, and avoid inactivation resulting from an excess H2O2. This report is the first of the Mn2+-independent production of an active versatile peroxidase by P. ostreatus. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Manganese peroxidase; White rot fungi; Gene expression; Enzyme stability
Ligninolytic peroxidases have been isolated and characterized from numerous white rot fungi and can directly oxidize a variety of substrates [1]. In Phanerochaete chrysosporium, lignin peroxidase (LiP) oxidizes a non-phenolic unit of lignin to produce a cation radical on the aromatic ring [2], whereas manganese peroxidase (MnP) has a Mn2+-binding site and oxidizes Mn2+ to Mn3+, in the presence of H2O2. The Mn3+ is then stabilized by organic chelators in the culture filtrates to oxidize phenolic substrates [3]. MnP withdraws one electron from phenolic compounds to generate a phenoxy radical [4], although it also oxidizes non-phenolics in combination with appropriate redox mediators or by initiating free radical chain reactions of unsaturated fatty acid [5,6]. LiP and MnP had been thought to represent two classes of fungal ligninolytic peroxidases, but *
Corresponding author. Fax: +81 774 38 3643. E-mail address: [email protected] (Y. Honda).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.084
many peroxidase enzymes which are difficult to classify as either LiP or MnP are also produced by other white rot basidiomycete fungi [7]. Notably, in Pleurotus species, several extracellular peroxidases with a Mn2+-binding site have been described. These enzymes can efficiently oxidize Mn2+ to Mn3+. Some of them also can utilize phenolic compounds to complete their catalytic cycles [7–9], and others can directly oxidize nonphenolic and/or high-molecular weight compounds and are known as hybrid-type or versatile peroxidases (VP) [8,10]. Three mnp genes, namely mnp1, mnp2, and mnp3, have been isolated and their products were characterized from Pleurotus ostreatus [11–13]. A partial cDNA fragment with similarity to Pleurotus eryngii vpl, detected in RT-PCR experiments, has been named mnp4 [14]. MnP2 and MnP3 can each be the dominant form depending on the culture medium [20]. Both MnP2 and MnP3 have been purified to homogeneity [9,12,15,16], although
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small additional peaks with manganese peroxidase activity were observed in an anion-exchange chromatography experiment with cultures grown on peptone media containing perlite [14]. Giardina et al. [12] reported that MnP2 from P. ostreatus (strain Florida) did not oxidize non-phenolic compounds such as veratryl alcohol. However, we found that a homologous enzyme, MnP-GY, from P. ostreatus (strain ATCC66376), could directly oxidize veratryl alcohol and high-molecular weight compounds, such as Poly R-478 and RNaseA, although much less efficiently than it oxidized Mn2+ [10,15]. We cloned a gene encoding MnP2 from the same strain [15] and showed that the deduced amino acid sequence was the same as that of MnP2 from strain Florida [12]. The difference in enzymatic properties between these two MnP2 isozymes might be due to differences in post-translational modification. The deduced amino acid sequence of P. ostreatus MnP2 was 96% identical to that of a typical VP, PS1 from P. eryngii [8]. A tryptophan residue conserved in all the VPs also was essential for the versatile peroxidase function of MnP2 from P. ostreatus strain ATCC66376, based on a chemical modification with N-bromosuccimide [10]. To date, analyses of P. ostreatus MnP expression have been made using various culture media containing natural nutrients, such as peptone, yeast extracts, wood meal, and cotton stalk [9,12,15,17]. These ‘‘natural’’ nutrients are complicated mixtures of various organic and inorganic materials. Although solid substrates mimic conditions for wood degradation better, a liquid medium is more suitable for monitoring the regulation of gene expression by nutrient components, and production and characterization of the enzymes. To make it clear which component regulates the enzyme production and by what mechanism the regulation occurs, a synthetic medium has an advantage over other media containing coexisting and non-preferable substrates. In this report, synthetic, chemically defined media were used to characterize the regulatory effect of nitrogen sources, Mn2+ and Poly R-478, on the expression of P. ostreatus MnP2.
Materials and methods Fungal cultures. Pleurotus ostreatus (ATCC66376) was used throughout this study. MnP was produced using a synthetic liquid medium containing 3% glucose, 1% Kirks salts (without MnSO4), 270 lM MnSO4, and 20 mM Na-phthalate buffer (pH 4.5) in addition to 0.5–10 mM ammonium oxalate as a nitrogen source. Kirks salts (without MnSO4) contained the following per liter of deionized water from EASYpure system (Barnstead/Thermolyne, Dubuque, IA, USA): KH2PO4, 2.0 g; MgSO4 Æ 7H2O, 0.5 g; CaCl2, 0.1 g; thiamine, 1 mg; and minerals solution, 10 ml. Minerals (per liter of deionized water) consisted of the following: nitrilotriacetic acid, 1.5 g; MgSO4 Æ 7H2O, 3.0 g; NaCl, 1.0 g; FeSO4 Æ 7H2O, 0.1 g; CoSO4, 0.1 g; AlK(SO4)2, 0.01 g; H3BO3, 0.01 g; and NaMoO4, 0.01 g [18]. A medium containing 3% glucose, 1% Kirks salts (without MnSO4), 1.0 mM ammonium
oxalate, and 20 mM Na-phthalate buffer (pH 4.5) was used as a basic medium for analyzing the transcript level of mnp2 with or without Mn2+. Poly R-478 (0.1%) was added to the basic medium containing 1.0 mM ammonium oxalate to monitor its effect on the accumulation of transcripts and enzyme activity in the low-nitrogen conditions. Four pieces of mycelial mats (6 mm /) grown on 3.9% potato–dextrose–agar (Nissui, Tokyo, Japan) plates at 28 °C for eight days were cut off and inoculated onto 30 ml of the medium in a 300-ml Erlenmeyer flask. The cultures were maintained statically at 28 °C in darkness. Assay of MnP activity. MnP activity was assayed using a reaction mixture containing 0.4 mM guaiacol, 50 mM Na-lactate buffer (pH 4.5), 0.2 mM MnSO4, and 0.1 mM H2O2 in a total volume of 1 ml [19]. The reaction was monitored by measuring the increase in absorbance of the reaction product at 465 nm. The level of activity was calculated by subtracting the value obtained in the absence from that in the presence of Mn2+. One unit of MnP activity was defined as the amount of enzyme that increased the absorbance at 465 nm by 1.0 per minute. Analysis of isozyme profiles of the extracellular MnP. The culture filtrate was dialyzed against a 20 mM Na-succinate buffer (pH 4.5) and 100 ml of dialyzate was concentrated to 700 ll by ultrafiltration using a Centriprep-30 microconcentrator (Millipore, Bedford, MA, USA). Then, 100 ll of concentrate was subjected to anion-exchange chromatography on a Mono-Q (5/5) column (Amersham Biosciences, Piscataway, NJ, USA). The isozymes were eluted with a linear gradient from 70 to 175 mM NaCl in 10 mM Na-succinate buffer (pH 5.0), over 120 min at a flow rate of 0.5 ml/min. The elution of the protein was monitored by measuring the absorbance at 407 nm specific to the heme protein and MnP activity was checked. The elution profile of each sample was compared to those of purified MnP2 and MnP3 isozymes. RT-PCR analyses of mnp2 transcript levels. RT-PCR amplification was performed using RNA samples from different culture conditions, in order to compare the relative abundance of mnp2 transcript and also b-tubulin gene transcripts as a control. Experimental condition and primer designs basically followed to those described by Cohen et al. [14] who evaluated the abundance of the four mnps separately. In the preliminary experiments, the condition described below was the best to monitor relative transcript abundance in our system (data not shown). Total RNA was extracted from mycelia grown on liquid medium for 13 days, unless otherwise stated. The mycelia, after a rapid wash with triple-distilled water, were filtered and quickly removed of moisture with filter paper. Samples were transferred to a 50-ml screw-capped tube containing 10 ml Isogen (Nippon Gene, Tokyo, Japan). Cells were disrupted with a polytron homogenizer PT1300D (Kinematica AG, Switzerland) and total RNA was extracted according to the protocol supplied for Isogen. RNA samples were stored at 80 °C prior to use. RT-PCRs were performed using the thermal cycler Personal (TaKaRa Bio, Kusatsu, Shiga, Japan) and a Ready-To-Go RTPCR Kit (Amersham Biosciences, Piscataway, NJ, USA). The reverse transcription was conducted with 0.5 lg total RNA and 0.5 lg oligo(dT)12–18 primer at 42 °C for 30 min, followed by 95 °C for 5 min to inactivate the reverse transcriptase. Then, 25 pmol of each specific primer was added for the PCR. The primers used were mnp2F (5 0 -ATC TTCAGTGACACCGAGCC-3 0 ) and mnp2R (5 0 -GGCGGTCCCTT GCAACATTGT-3 0 ), and b-tubF (5 0 -GTGCGTAAGGAAGCTG AGGG-3 0 ) and b-tubR (5 0 -CGGAATTCYTGRTAYTGYTGRTA YTC-3 0 ). The amplification program was: 95 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, and a final 9 min extension at 72 °C. After amplification, a 5-ll aliquot of each PCR product was analyzed by electrophoresis on an agarose gel. Distinct DNA bands of the expected size, 422 and 900 bp, were observed for mnp2 and b-tub cDNA fragments, respectively, and no other bands could be seen, indicating that no genomic DNA was contaminated (data not shown). Enzyme stability assay. MnP2 was purified to homogeneity as described previously [15]. Two unit of purified MnP2 in 1 ml of 50 mM lactate buffer (pH 4.5) was incubated at 25 °C with 0.1 mM H2O2, 0.4 mM MnSO4 or 0.4% Poly R-478, as indicated. A 50-ll aliquot was
H. Kamitsuji et al. / Biochemical and Biophysical Research Communications 327 (2005) 871–876 removed at 0, 0.5, 1, 1.5, 2, 4, 6, and 12 h, and the residual MnP activity was measured as described above. Relative MnP activity was evaluated with each value at the starting point taken as 100%.
Results and discussion Production of P. ostreatus MnP2 on chemically defined media To analyze the effects of nutrient nitrogen on MnP production, enzyme activity in the culture filtrate was measured when P. ostreatus was inoculated on synthetic media composed of 3% glucose, 270 lM MnSO4, and other minerals in 20 mM Na-phthalate buffer (see Materials and methods), supplemented with 0.5, 1, 2, and
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10 mM ammonium oxalate (Fig. 1A). At each concentration, MnP activity was detected from 14 days after the inoculation and increased continuously until day 25. At lower NHþ 4 concentrations, higher MnP activity was detected; the highest enzyme activity was observed when 1 mM ammonium oxalate was added to the medium. The growth rate of the mycelium was slightly higher in the high-NHþ 4 condition, as the dry weight of the mycelium harvested on day 14 from cultures containing 0.5, 1, 2, and 10 mM ammonium oxalate was 307, 312, 368, and 359 mg, respectively. Analysis of the isozyme profile with anion-exchange chromatography (Fig. 1B) demonstrated that MnP2 was predominantly expressed and a little amount of MnP3 was observed in these conditions. The predominant expression of MnP2 was confirmed with the following IEF analysis and N-terminal amino acid determination of the purified enzyme (data not shown). To monitor transcriptional regulation, RT-PCR analysis of mnp2 transcripts was performed (Fig. 1C). Large amounts of the mnp2 transcripts were observed at 0.5 and 1 mM NHþ 4 (lanes 1 and 2), but small and negligible amounts were detected at 2 and 10 mM NHþ 4 (lanes 3 and 4), respectively, whereas the level of b-tubulin transcript remained constant. The abundance of mnp2 transcripts was in good agreement with the MnP2 detected in the isozyme profile analysis at the corresponding NHþ 4 concentration (Fig. 1B). A similar down-regulation in MnP2 production and mnp2 transcription was observed when arginine or urea was used as a nitrogen source, instead of ammonium oxalate (data not shown). From these results, it was suggested that MnP2 expression was down-regulated by nutrient nitrogen, mostly at the transcriptional level. Effect of Mn2+ on MnP2 production
Fig. 1. Expression of P. ostreatus MnP in liquid culture using synthetic medium containing 0.5, 1, 2 or 10 mM ammonium oxalate. (A) Time course of MnP activity in the culture filtrate, (B) isozyme profile analysis with Mono-Q 5/5 column chromatography of culture filtrates on day 19, and (C) RT-PCR analysis of the transcript level of mnp2 on day 13 in the presence of 0.5 (lane 1), 1 (lane 2), 2 (lane 3) or 10 (lane 4) mM NHþ 4 was indicated. Transcript level of b-tub was also analyzed at each point as a control. The control in (B) shows an elution profile for the mixture of purified MnP2 and MnP3 isozymes.
It was reported that addition of Mn2+ resulted in significant changes in MnP activity secreted by P. ostreatus [12,14,17]. Differential effects on the transcript accumulation of the four mnps by Mn2+ added to a culture medium containing peptone or cotton stalk were observed [14,17]. As for transcription of mnp2, amendment of Mn2+ resulted in a down- and up-regulation when peptone- and cotton stalk-containing media were used, respectively [14,17]. In order to clearly understand the regulatory effect of Mn2+ on MnP2 production, P. ostreatus (ATCC66376) was inoculated on the synthetic medium without Mn2+ (see Materials and methods) and incubated at 28 °C for 20 days, then 270 lM MnSO4 was added to the culture. MnP activity was negligible throughout the culture period in the absence of Mn2+ (Fig. 2A). Addition of MnSO4 enabled detection of MnP activity extracellularly. Isozyme profile analysis with anion-exchange chromatography showed predominant production of MnP2 in the culture filtrate (data not shown). An SDS–polyacrylamide gel electrophoresis
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Effect of Poly R-478 on MnP2 production The effect of Poly R-478, a high-molecular weight substrate of MnP2 [10], on MnP expression was monitored in the following experiments. P. ostreatus was grown in low-nitrogen condition in the absence of Mn2+, with or without 0.1% Poly R-478. A significant MnP activity was observed when Poly R-478 was added to the medium (Fig. 3A). An anion-exchange chromatography experiment demonstrated a predominance of MnP2 in the culture filtrate (Fig. 3B). The following IEF analysis and determination of N-terminal amino acid sequence confirmed the production of MnP2 (data not shown). Similar Poly R-478-dependent detection of MnP activity was observed in a series of time-course assays using medium containing 0.001, 0.01, 0.05, and 0.5% Poly R-478 (data not shown). In these conditions, a larger amount of MnP activity was observed as the concentration of Poly R-478 increased: the highest level of MnP activity was observed in the medium containing Fig. 2. Effect of Mn2+ on extracellular MnP activity and mnp2 transcript level. P. ostreatus was cultivated in the absence of Mn2+ in the synthetic medium containing 1 mM ammonium oxalate for 20 days. Then MnSO4 was added at 270 lM to the culture. (A) Time course of MnP activity in the culture filtrate in the presence (closed circle) or absence (open circle) of Mn2+. (B) RT-PCR analysis of the transcript levels of mnp2, and b-tub as a control, was performed using RNA extracted from mycelium at 24 h (lane 2) and seven days (lane 4) after addition of Mn2+. Similar experiments without addition of Mn2+ were performed at 24 h (lane 1) and seven days (lane 3).
analysis of the culture filtrate demonstrated that a distinct protein band co-migrating with purified MnP2 was observed in Mn2+-sufficient but not Mn2+-deficient cultures (data not shown). The effect of Mn2+ on the level of mnp2 transcripts was analyzed by RT-PCR experiments at 24 h and seven days after the addition of MnSO4 (Fig. 2B). In the absence of Mn2+, accumulation of the mnp2 transcripts was observed at both sampling times (lanes 1 and 3). These results clearly indicated that Mn2+ is dispensable for the transcription of mnp2 using a chemically defined synthetic medium. Addition of MnSO4 resulted in a slight decrease in mnp2 transcript levels after 24 h (lane 2), which is consistent with the results observed with peptone-containing medium [14]. However, differences in mnp2 transcript levels were negligible in Mn2+-deficient and sufficient cultures at day 27 (lane 4). It has been proposed that an Mn2+-dependent posttranscriptional process is essential for MnP production in white rot fungi such as P. chrysosporium [20] and Ceriporiopsis subvermispora [21], because Mn2+ is indispensably required for the production of active MnP but not for the accumulation of the transcripts. Our data also suggested that Mn2+ may play a significant role in a step between transcription and accumulation of active MnP2 by P. ostreatus.
Fig. 3. Effect of Poly R-478 on extracellular MnP activity and mnp2 transcript level. (A) Time course of MnP activity in the culture filtrate in the presence (closed circle) or absence (open circle) of 0.1% Poly R478, (B) isozyme profile analysis with Mono-Q 5/5 column chromatography of culture filtrate on day 23 in the presence of Poly R-478, and (C) RT-PCR analysis of the transcript levels of mnp2, and b-tub as a control, at indicated incubation periods was demonstrated. The control in (B) shows an elution profile for the purified MnP2.
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0.5% Poly R-478 (1.24 U/ml on day 35). Interestingly, RT-PCR analysis of messenger RNA revealed that the mnp2 transcription was not affected by Poly R-478 in the medium (Fig. 3C), indicating that Poly R-478 does not act as a transcriptional inducer. Suppression of H2O2-induced enzyme inactivation by Poly R-478 As shown in Fig. 1, Poly R-478 was not essential for MnP2 production in the presence of Mn2+. To elucidate the mechanism for the Poly R-478-dependent accumulation of MnP2 in the absence of Mn2+, we checked whether the substrate can be used to avoid enzyme inactivation caused by the bleaching of heme via compound III formation by H2O2. When purified MnP2 was exposed to an excess H2O2, the activity was lost rapidly (Fig. 4). However, in the presence of Mn2+, this H2O2induced inactivation did not take place (Fig. 4A), indicating that formation of compound III was prevented as the catalytic cycle was completed by oxidizing Mn2+. We reported that Mn2+ acts as a dissociation
Fig. 4. Stabilization of MnP2 by Mn2+ or Poly R-478 against H2O2induced inactivation. Purified MnP2 was exposed to 0.1 mM H2O2 in 20 mM lactate buffer (pH 4.5), in the presence (closed circle) or absence (closed square) of Mn2+ (A), or Poly R-478 (B), and the remaining MnP activity was periodically measured. Enzyme activity in the condition of MnP2 alone (open square), and MnP2 with Mn2+ or Poly R-478 (open circle) was also analyzed in the absence of H2O2. The remaining MnP activity was evaluated with each value at the starting point taken as 100%.
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agent of O2 from compound III of P. ostreatus MnP2 [22]. When Poly R-478 was added to the mixture, the H2O2-induced inactivation was avoided (Fig. 4B). The Poly R-478-dependent enzyme protection was not observed, while Mn2+ protected the enzyme from inactivation, when MnP3 was exposed to an excess H2O2 in similar experiments (data not shown). From the results presented above, it seemed that Mn2+ or Poly R-478 was required for MnP2 production as a direct substrate to avoid inactivation of the enzyme by H2O2 in the culture medium. We tried to measure the H2O2 concentration in the culture filtrate but were unsuccessful, possibly because H2O2 is easily reduced and used up by the coexisting MnP. However, white rot fungi themselves have H2O2-producing system [23– 25] and we detected anisyl alcohol oxidase activity in the culture filtrate in these experiments (data not shown). H2O2-induced inactivation of VP and stabilization by the addition of Mn2+ or phenolic compounds were also reported in Pleurotus pulmonarius [26]. In the present study, we suggested that the dependence of MnP2 production on Mn2+ results from stabilization of the enzyme to avoid H2O2-induced inactivation in the culture filtrate. It was also demonstrated that a high-molecular weight substrate of MnP2, Poly R-478 can substitute for Mn2+ in MnP2 production by P. ostreatus. So in the case of MnP2, there is no evidence that an essential Mn2+-dependent post-transcriptional process actually exists. It has also been reported that Mn2+ is dispensable for LiP production in P. chrysosporium [27]. Generally, it is difficult to demonstrate for manganese-dependent peroxidases whether the essential requirement for Mn2+ results from an indispensable role, if any, in the expression pathway or merely a contribution to enzyme stabilization in the culture medium. However, one must be very careful when discussing the Mn2+-dependent post-transcriptional process in white rot fungi, because ligninolytic peroxidases, including manganese-dependent ones, are heterologously produced in ascomycetous fungi [28,29] and even in Escherichia coli [30–32]. Moreover, it is conceivable that, among peroxidases with a Mn-binding site, those with the conserved tryptophane (Trp170 in MnP2) can be classified as versatile peroxidases and the others as classical manganese-dependent peroxidases [1,4]. In this context, it is not plausible that Mn2+ plays an essential rule in the post-transcriptional process exclusively for the production of manganese-dependent peroxidases in white rot fungi.
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Mn2+ is dispensable for the production of active MnP2 by Pleurotus ostreatus Hisatoshi Kamitsujia, Yoichi Hondaa,*, Takashi Watanabea, Masaaki Kuwaharab a
Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan Institute of Wood Technology, Akita Prefectural University, Kaieizaka, Noshiro, Akita 016-0876, Japan
b
Received 30 November 2004 Available online 24 December 2004
Abstract The regulation mechanism for expression of versatile peroxidase MnP2 by the basidiomycete fungus Pleurotus ostreatus was examined using chemically defined synthetic media. Expression of MnP2 was down-regulated at the transcription level by nutrient nitrogen, e.g., NHþ 4 , arginine or urea. As is often the case with other fungal manganese peroxidases, active MnP2 was not detected when Mn2+ was omitted from the culture, while mnp2 transcription was barely affected by Mn2+. However, Mn2+ can be substituted by an MnP2 substrate, Poly R-478, since active MnP2 was detected extracellularly when the compound was added to the culture without Mn2+. Enzyme stability assays with the purified MnP2 indicated an indispensable requirement for a substrate that can be used to complete the catalytic cycle, and avoid inactivation resulting from an excess H2O2. This report is the first of the Mn2+-independent production of an active versatile peroxidase by P. ostreatus. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Manganese peroxidase; White rot fungi; Gene expression; Enzyme stability
Ligninolytic peroxidases have been isolated and characterized from numerous white rot fungi and can directly oxidize a variety of substrates [1]. In Phanerochaete chrysosporium, lignin peroxidase (LiP) oxidizes a non-phenolic unit of lignin to produce a cation radical on the aromatic ring [2], whereas manganese peroxidase (MnP) has a Mn2+-binding site and oxidizes Mn2+ to Mn3+, in the presence of H2O2. The Mn3+ is then stabilized by organic chelators in the culture filtrates to oxidize phenolic substrates [3]. MnP withdraws one electron from phenolic compounds to generate a phenoxy radical [4], although it also oxidizes non-phenolics in combination with appropriate redox mediators or by initiating free radical chain reactions of unsaturated fatty acid [5,6]. LiP and MnP had been thought to represent two classes of fungal ligninolytic peroxidases, but *
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0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.084
many peroxidase enzymes which are difficult to classify as either LiP or MnP are also produced by other white rot basidiomycete fungi [7]. Notably, in Pleurotus species, several extracellular peroxidases with a Mn2+-binding site have been described. These enzymes can efficiently oxidize Mn2+ to Mn3+. Some of them also can utilize phenolic compounds to complete their catalytic cycles [7–9], and others can directly oxidize nonphenolic and/or high-molecular weight compounds and are known as hybrid-type or versatile peroxidases (VP) [8,10]. Three mnp genes, namely mnp1, mnp2, and mnp3, have been isolated and their products were characterized from Pleurotus ostreatus [11–13]. A partial cDNA fragment with similarity to Pleurotus eryngii vpl, detected in RT-PCR experiments, has been named mnp4 [14]. MnP2 and MnP3 can each be the dominant form depending on the culture medium [20]. Both MnP2 and MnP3 have been purified to homogeneity [9,12,15,16], although
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small additional peaks with manganese peroxidase activity were observed in an anion-exchange chromatography experiment with cultures grown on peptone media containing perlite [14]. Giardina et al. [12] reported that MnP2 from P. ostreatus (strain Florida) did not oxidize non-phenolic compounds such as veratryl alcohol. However, we found that a homologous enzyme, MnP-GY, from P. ostreatus (strain ATCC66376), could directly oxidize veratryl alcohol and high-molecular weight compounds, such as Poly R-478 and RNaseA, although much less efficiently than it oxidized Mn2+ [10,15]. We cloned a gene encoding MnP2 from the same strain [15] and showed that the deduced amino acid sequence was the same as that of MnP2 from strain Florida [12]. The difference in enzymatic properties between these two MnP2 isozymes might be due to differences in post-translational modification. The deduced amino acid sequence of P. ostreatus MnP2 was 96% identical to that of a typical VP, PS1 from P. eryngii [8]. A tryptophan residue conserved in all the VPs also was essential for the versatile peroxidase function of MnP2 from P. ostreatus strain ATCC66376, based on a chemical modification with N-bromosuccimide [10]. To date, analyses of P. ostreatus MnP expression have been made using various culture media containing natural nutrients, such as peptone, yeast extracts, wood meal, and cotton stalk [9,12,15,17]. These ‘‘natural’’ nutrients are complicated mixtures of various organic and inorganic materials. Although solid substrates mimic conditions for wood degradation better, a liquid medium is more suitable for monitoring the regulation of gene expression by nutrient components, and production and characterization of the enzymes. To make it clear which component regulates the enzyme production and by what mechanism the regulation occurs, a synthetic medium has an advantage over other media containing coexisting and non-preferable substrates. In this report, synthetic, chemically defined media were used to characterize the regulatory effect of nitrogen sources, Mn2+ and Poly R-478, on the expression of P. ostreatus MnP2.
Materials and methods Fungal cultures. Pleurotus ostreatus (ATCC66376) was used throughout this study. MnP was produced using a synthetic liquid medium containing 3% glucose, 1% Kirks salts (without MnSO4), 270 lM MnSO4, and 20 mM Na-phthalate buffer (pH 4.5) in addition to 0.5–10 mM ammonium oxalate as a nitrogen source. Kirks salts (without MnSO4) contained the following per liter of deionized water from EASYpure system (Barnstead/Thermolyne, Dubuque, IA, USA): KH2PO4, 2.0 g; MgSO4 Æ 7H2O, 0.5 g; CaCl2, 0.1 g; thiamine, 1 mg; and minerals solution, 10 ml. Minerals (per liter of deionized water) consisted of the following: nitrilotriacetic acid, 1.5 g; MgSO4 Æ 7H2O, 3.0 g; NaCl, 1.0 g; FeSO4 Æ 7H2O, 0.1 g; CoSO4, 0.1 g; AlK(SO4)2, 0.01 g; H3BO3, 0.01 g; and NaMoO4, 0.01 g [18]. A medium containing 3% glucose, 1% Kirks salts (without MnSO4), 1.0 mM ammonium
oxalate, and 20 mM Na-phthalate buffer (pH 4.5) was used as a basic medium for analyzing the transcript level of mnp2 with or without Mn2+. Poly R-478 (0.1%) was added to the basic medium containing 1.0 mM ammonium oxalate to monitor its effect on the accumulation of transcripts and enzyme activity in the low-nitrogen conditions. Four pieces of mycelial mats (6 mm /) grown on 3.9% potato–dextrose–agar (Nissui, Tokyo, Japan) plates at 28 °C for eight days were cut off and inoculated onto 30 ml of the medium in a 300-ml Erlenmeyer flask. The cultures were maintained statically at 28 °C in darkness. Assay of MnP activity. MnP activity was assayed using a reaction mixture containing 0.4 mM guaiacol, 50 mM Na-lactate buffer (pH 4.5), 0.2 mM MnSO4, and 0.1 mM H2O2 in a total volume of 1 ml [19]. The reaction was monitored by measuring the increase in absorbance of the reaction product at 465 nm. The level of activity was calculated by subtracting the value obtained in the absence from that in the presence of Mn2+. One unit of MnP activity was defined as the amount of enzyme that increased the absorbance at 465 nm by 1.0 per minute. Analysis of isozyme profiles of the extracellular MnP. The culture filtrate was dialyzed against a 20 mM Na-succinate buffer (pH 4.5) and 100 ml of dialyzate was concentrated to 700 ll by ultrafiltration using a Centriprep-30 microconcentrator (Millipore, Bedford, MA, USA). Then, 100 ll of concentrate was subjected to anion-exchange chromatography on a Mono-Q (5/5) column (Amersham Biosciences, Piscataway, NJ, USA). The isozymes were eluted with a linear gradient from 70 to 175 mM NaCl in 10 mM Na-succinate buffer (pH 5.0), over 120 min at a flow rate of 0.5 ml/min. The elution of the protein was monitored by measuring the absorbance at 407 nm specific to the heme protein and MnP activity was checked. The elution profile of each sample was compared to those of purified MnP2 and MnP3 isozymes. RT-PCR analyses of mnp2 transcript levels. RT-PCR amplification was performed using RNA samples from different culture conditions, in order to compare the relative abundance of mnp2 transcript and also b-tubulin gene transcripts as a control. Experimental condition and primer designs basically followed to those described by Cohen et al. [14] who evaluated the abundance of the four mnps separately. In the preliminary experiments, the condition described below was the best to monitor relative transcript abundance in our system (data not shown). Total RNA was extracted from mycelia grown on liquid medium for 13 days, unless otherwise stated. The mycelia, after a rapid wash with triple-distilled water, were filtered and quickly removed of moisture with filter paper. Samples were transferred to a 50-ml screw-capped tube containing 10 ml Isogen (Nippon Gene, Tokyo, Japan). Cells were disrupted with a polytron homogenizer PT1300D (Kinematica AG, Switzerland) and total RNA was extracted according to the protocol supplied for Isogen. RNA samples were stored at 80 °C prior to use. RT-PCRs were performed using the thermal cycler Personal (TaKaRa Bio, Kusatsu, Shiga, Japan) and a Ready-To-Go RTPCR Kit (Amersham Biosciences, Piscataway, NJ, USA). The reverse transcription was conducted with 0.5 lg total RNA and 0.5 lg oligo(dT)12–18 primer at 42 °C for 30 min, followed by 95 °C for 5 min to inactivate the reverse transcriptase. Then, 25 pmol of each specific primer was added for the PCR. The primers used were mnp2F (5 0 -ATC TTCAGTGACACCGAGCC-3 0 ) and mnp2R (5 0 -GGCGGTCCCTT GCAACATTGT-3 0 ), and b-tubF (5 0 -GTGCGTAAGGAAGCTG AGGG-3 0 ) and b-tubR (5 0 -CGGAATTCYTGRTAYTGYTGRTA YTC-3 0 ). The amplification program was: 95 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, and a final 9 min extension at 72 °C. After amplification, a 5-ll aliquot of each PCR product was analyzed by electrophoresis on an agarose gel. Distinct DNA bands of the expected size, 422 and 900 bp, were observed for mnp2 and b-tub cDNA fragments, respectively, and no other bands could be seen, indicating that no genomic DNA was contaminated (data not shown). Enzyme stability assay. MnP2 was purified to homogeneity as described previously [15]. Two unit of purified MnP2 in 1 ml of 50 mM lactate buffer (pH 4.5) was incubated at 25 °C with 0.1 mM H2O2, 0.4 mM MnSO4 or 0.4% Poly R-478, as indicated. A 50-ll aliquot was
H. Kamitsuji et al. / Biochemical and Biophysical Research Communications 327 (2005) 871–876 removed at 0, 0.5, 1, 1.5, 2, 4, 6, and 12 h, and the residual MnP activity was measured as described above. Relative MnP activity was evaluated with each value at the starting point taken as 100%.
Results and discussion Production of P. ostreatus MnP2 on chemically defined media To analyze the effects of nutrient nitrogen on MnP production, enzyme activity in the culture filtrate was measured when P. ostreatus was inoculated on synthetic media composed of 3% glucose, 270 lM MnSO4, and other minerals in 20 mM Na-phthalate buffer (see Materials and methods), supplemented with 0.5, 1, 2, and
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10 mM ammonium oxalate (Fig. 1A). At each concentration, MnP activity was detected from 14 days after the inoculation and increased continuously until day 25. At lower NHþ 4 concentrations, higher MnP activity was detected; the highest enzyme activity was observed when 1 mM ammonium oxalate was added to the medium. The growth rate of the mycelium was slightly higher in the high-NHþ 4 condition, as the dry weight of the mycelium harvested on day 14 from cultures containing 0.5, 1, 2, and 10 mM ammonium oxalate was 307, 312, 368, and 359 mg, respectively. Analysis of the isozyme profile with anion-exchange chromatography (Fig. 1B) demonstrated that MnP2 was predominantly expressed and a little amount of MnP3 was observed in these conditions. The predominant expression of MnP2 was confirmed with the following IEF analysis and N-terminal amino acid determination of the purified enzyme (data not shown). To monitor transcriptional regulation, RT-PCR analysis of mnp2 transcripts was performed (Fig. 1C). Large amounts of the mnp2 transcripts were observed at 0.5 and 1 mM NHþ 4 (lanes 1 and 2), but small and negligible amounts were detected at 2 and 10 mM NHþ 4 (lanes 3 and 4), respectively, whereas the level of b-tubulin transcript remained constant. The abundance of mnp2 transcripts was in good agreement with the MnP2 detected in the isozyme profile analysis at the corresponding NHþ 4 concentration (Fig. 1B). A similar down-regulation in MnP2 production and mnp2 transcription was observed when arginine or urea was used as a nitrogen source, instead of ammonium oxalate (data not shown). From these results, it was suggested that MnP2 expression was down-regulated by nutrient nitrogen, mostly at the transcriptional level. Effect of Mn2+ on MnP2 production
Fig. 1. Expression of P. ostreatus MnP in liquid culture using synthetic medium containing 0.5, 1, 2 or 10 mM ammonium oxalate. (A) Time course of MnP activity in the culture filtrate, (B) isozyme profile analysis with Mono-Q 5/5 column chromatography of culture filtrates on day 19, and (C) RT-PCR analysis of the transcript level of mnp2 on day 13 in the presence of 0.5 (lane 1), 1 (lane 2), 2 (lane 3) or 10 (lane 4) mM NHþ 4 was indicated. Transcript level of b-tub was also analyzed at each point as a control. The control in (B) shows an elution profile for the mixture of purified MnP2 and MnP3 isozymes.
It was reported that addition of Mn2+ resulted in significant changes in MnP activity secreted by P. ostreatus [12,14,17]. Differential effects on the transcript accumulation of the four mnps by Mn2+ added to a culture medium containing peptone or cotton stalk were observed [14,17]. As for transcription of mnp2, amendment of Mn2+ resulted in a down- and up-regulation when peptone- and cotton stalk-containing media were used, respectively [14,17]. In order to clearly understand the regulatory effect of Mn2+ on MnP2 production, P. ostreatus (ATCC66376) was inoculated on the synthetic medium without Mn2+ (see Materials and methods) and incubated at 28 °C for 20 days, then 270 lM MnSO4 was added to the culture. MnP activity was negligible throughout the culture period in the absence of Mn2+ (Fig. 2A). Addition of MnSO4 enabled detection of MnP activity extracellularly. Isozyme profile analysis with anion-exchange chromatography showed predominant production of MnP2 in the culture filtrate (data not shown). An SDS–polyacrylamide gel electrophoresis
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Effect of Poly R-478 on MnP2 production The effect of Poly R-478, a high-molecular weight substrate of MnP2 [10], on MnP expression was monitored in the following experiments. P. ostreatus was grown in low-nitrogen condition in the absence of Mn2+, with or without 0.1% Poly R-478. A significant MnP activity was observed when Poly R-478 was added to the medium (Fig. 3A). An anion-exchange chromatography experiment demonstrated a predominance of MnP2 in the culture filtrate (Fig. 3B). The following IEF analysis and determination of N-terminal amino acid sequence confirmed the production of MnP2 (data not shown). Similar Poly R-478-dependent detection of MnP activity was observed in a series of time-course assays using medium containing 0.001, 0.01, 0.05, and 0.5% Poly R-478 (data not shown). In these conditions, a larger amount of MnP activity was observed as the concentration of Poly R-478 increased: the highest level of MnP activity was observed in the medium containing Fig. 2. Effect of Mn2+ on extracellular MnP activity and mnp2 transcript level. P. ostreatus was cultivated in the absence of Mn2+ in the synthetic medium containing 1 mM ammonium oxalate for 20 days. Then MnSO4 was added at 270 lM to the culture. (A) Time course of MnP activity in the culture filtrate in the presence (closed circle) or absence (open circle) of Mn2+. (B) RT-PCR analysis of the transcript levels of mnp2, and b-tub as a control, was performed using RNA extracted from mycelium at 24 h (lane 2) and seven days (lane 4) after addition of Mn2+. Similar experiments without addition of Mn2+ were performed at 24 h (lane 1) and seven days (lane 3).
analysis of the culture filtrate demonstrated that a distinct protein band co-migrating with purified MnP2 was observed in Mn2+-sufficient but not Mn2+-deficient cultures (data not shown). The effect of Mn2+ on the level of mnp2 transcripts was analyzed by RT-PCR experiments at 24 h and seven days after the addition of MnSO4 (Fig. 2B). In the absence of Mn2+, accumulation of the mnp2 transcripts was observed at both sampling times (lanes 1 and 3). These results clearly indicated that Mn2+ is dispensable for the transcription of mnp2 using a chemically defined synthetic medium. Addition of MnSO4 resulted in a slight decrease in mnp2 transcript levels after 24 h (lane 2), which is consistent with the results observed with peptone-containing medium [14]. However, differences in mnp2 transcript levels were negligible in Mn2+-deficient and sufficient cultures at day 27 (lane 4). It has been proposed that an Mn2+-dependent posttranscriptional process is essential for MnP production in white rot fungi such as P. chrysosporium [20] and Ceriporiopsis subvermispora [21], because Mn2+ is indispensably required for the production of active MnP but not for the accumulation of the transcripts. Our data also suggested that Mn2+ may play a significant role in a step between transcription and accumulation of active MnP2 by P. ostreatus.
Fig. 3. Effect of Poly R-478 on extracellular MnP activity and mnp2 transcript level. (A) Time course of MnP activity in the culture filtrate in the presence (closed circle) or absence (open circle) of 0.1% Poly R478, (B) isozyme profile analysis with Mono-Q 5/5 column chromatography of culture filtrate on day 23 in the presence of Poly R-478, and (C) RT-PCR analysis of the transcript levels of mnp2, and b-tub as a control, at indicated incubation periods was demonstrated. The control in (B) shows an elution profile for the purified MnP2.
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0.5% Poly R-478 (1.24 U/ml on day 35). Interestingly, RT-PCR analysis of messenger RNA revealed that the mnp2 transcription was not affected by Poly R-478 in the medium (Fig. 3C), indicating that Poly R-478 does not act as a transcriptional inducer. Suppression of H2O2-induced enzyme inactivation by Poly R-478 As shown in Fig. 1, Poly R-478 was not essential for MnP2 production in the presence of Mn2+. To elucidate the mechanism for the Poly R-478-dependent accumulation of MnP2 in the absence of Mn2+, we checked whether the substrate can be used to avoid enzyme inactivation caused by the bleaching of heme via compound III formation by H2O2. When purified MnP2 was exposed to an excess H2O2, the activity was lost rapidly (Fig. 4). However, in the presence of Mn2+, this H2O2induced inactivation did not take place (Fig. 4A), indicating that formation of compound III was prevented as the catalytic cycle was completed by oxidizing Mn2+. We reported that Mn2+ acts as a dissociation
Fig. 4. Stabilization of MnP2 by Mn2+ or Poly R-478 against H2O2induced inactivation. Purified MnP2 was exposed to 0.1 mM H2O2 in 20 mM lactate buffer (pH 4.5), in the presence (closed circle) or absence (closed square) of Mn2+ (A), or Poly R-478 (B), and the remaining MnP activity was periodically measured. Enzyme activity in the condition of MnP2 alone (open square), and MnP2 with Mn2+ or Poly R-478 (open circle) was also analyzed in the absence of H2O2. The remaining MnP activity was evaluated with each value at the starting point taken as 100%.
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agent of O2 from compound III of P. ostreatus MnP2 [22]. When Poly R-478 was added to the mixture, the H2O2-induced inactivation was avoided (Fig. 4B). The Poly R-478-dependent enzyme protection was not observed, while Mn2+ protected the enzyme from inactivation, when MnP3 was exposed to an excess H2O2 in similar experiments (data not shown). From the results presented above, it seemed that Mn2+ or Poly R-478 was required for MnP2 production as a direct substrate to avoid inactivation of the enzyme by H2O2 in the culture medium. We tried to measure the H2O2 concentration in the culture filtrate but were unsuccessful, possibly because H2O2 is easily reduced and used up by the coexisting MnP. However, white rot fungi themselves have H2O2-producing system [23– 25] and we detected anisyl alcohol oxidase activity in the culture filtrate in these experiments (data not shown). H2O2-induced inactivation of VP and stabilization by the addition of Mn2+ or phenolic compounds were also reported in Pleurotus pulmonarius [26]. In the present study, we suggested that the dependence of MnP2 production on Mn2+ results from stabilization of the enzyme to avoid H2O2-induced inactivation in the culture filtrate. It was also demonstrated that a high-molecular weight substrate of MnP2, Poly R-478 can substitute for Mn2+ in MnP2 production by P. ostreatus. So in the case of MnP2, there is no evidence that an essential Mn2+-dependent post-transcriptional process actually exists. It has also been reported that Mn2+ is dispensable for LiP production in P. chrysosporium [27]. Generally, it is difficult to demonstrate for manganese-dependent peroxidases whether the essential requirement for Mn2+ results from an indispensable role, if any, in the expression pathway or merely a contribution to enzyme stabilization in the culture medium. However, one must be very careful when discussing the Mn2+-dependent post-transcriptional process in white rot fungi, because ligninolytic peroxidases, including manganese-dependent ones, are heterologously produced in ascomycetous fungi [28,29] and even in Escherichia coli [30–32]. Moreover, it is conceivable that, among peroxidases with a Mn-binding site, those with the conserved tryptophane (Trp170 in MnP2) can be classified as versatile peroxidases and the others as classical manganese-dependent peroxidases [1,4]. In this context, it is not plausible that Mn2+ plays an essential rule in the post-transcriptional process exclusively for the production of manganese-dependent peroxidases in white rot fungi.
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