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Molecular characterization of cytochrome P450 catalyzing hydroxylation of benzoates from the white-rot fungus Phanerochaete chrysosporium
BBRC Biochemical and Biophysical Research Communications 334 (2005) 1184–1190 www.elsevier.com/locate/ybbrc
Molecular characterization of cytochrome P450 catalyzing hydroxylation of benzoates from the white-rot fungus Phanerochaete chrysosporium Fumiko Matsuzaki, Hiroyuki Wariishi * Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan Received 1 July 2005 Available online 18 July 2005
Abstract We cloned full-length cDNA (PcCYP1f) encoding one of the cytochrome P450s in the lignin-degrading basidiomycete Phanerochaete chrysosporium, which showed high homology to P450s in the CYP53 family. PcCYP1f was expressed as an active microsomal protein using the methylotrophic yeast Pichia pastoris expression system. Using the microsomal fraction containing PcCYP1f, a typical P450 CO-difference spectrum was obtained with absorption maximum at 448 nm. Recombinant PcCYP1f catalyzed the hydroxylation of benzoic acid into 4-hydroxybenzoic acid in the presence of NADPH and P. chrysosporium cytochrome P450 oxidoreductase. In contrast to other CYP53 P450s, this enzyme was shown to catalyze the hydroxylation of 3-hydroxybenzoate into 3,4-dihydroxybenzoate. Furthermore, 2- and 3-methylbenzoate were also shown to be substrates of PcCYP1f. This is the first report showing the expression of a functionally active Phanerochaete P450. Finally, real-time quantitative PCR analysis revealed that PcCYP1f is induced at a transcriptional level by exogenous addition of benzoic acid. 2005 Elsevier Inc. All rights reserved. Keywords: Basidiomycete; Benzoate hydroxylase; Carbon monoxide-difference spectrum; Cytochrome P450; Heterologous expression; Kinetic analysis; Phanerochaete chrysosporium; Pichia pastoris; Real-time PCR; Substrate binding spectrum
White-rot fungi are capable of degrading a wide variety of recalcitrant aromatic compounds, including polymeric lignin and environmentally persistent pollutants. Extracellular ligninolytic enzymes, such as lignin and manganese peroxidases, involved in the metabolism of aromatic compounds have been extensively studied [1,2]. Intracellularly, cytochrome P450 (P450)-mediated oxygenation reactions are known to play an important role during fungal metabolism of recalcitrant xenobiotic compounds [3–10]. Phanerochaete chrysosporium is the most extensively studied white-rot fungus with regard to ligninolysis and xenobiotic metabolism. The gene diversity of fungal P450 was recently suggested via the whole sequence of the P. chrysosporium genome, where *
Corresponding author. Fax: +81 92 642 2992. E-mail address: [email protected] (H. Wariishi).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.013
as many as 148 P450 genes have been found [11]. Recently, we utilized a series of well-characterized P450 substrates as metabolic substrates for P. chrysosporium, pointing out not only the gene diversity but also the functional diversity of fungal P450 molecular species. Among the substrates examined, benzoate was found to be converted by Phanerochaete P450(s) [12]. In the present study, we cloned PcCYP1f, one of the Phanerochaete P450 genes, and showed high homology to the P450s in the CYP53 family (benzoate-4-hydroxylase), which are known to catalyze the hydroxylation of benzoic acid at the 4-position [13–15]. PcCYP1f was expressed in the heterologous expression system using Pichia pastoris. In the CO-difference spectrum, recombinant PcCYP1f exhibited absorption maximum at 448 nm with no contamination of absorption at 420 nm. It was also shown to catalyze the hydroxylation of benzoic acid
F. Matsuzaki, H. Wariishi / Biochemical and Biophysical Research Communications 334 (2005) 1184–1190
into 4-hydroxybenzoic acid in the presence of NADPH and P. chrysosporium cytochrome P450 reductase (PcCPR). Finally, real-time quantitative PCR analysis revealed that PcCYP1f was induced at the transcriptional level by exogenous addition of benzoic acid.
Materials and methods Chemicals. Benzoic acid was purchased from Wako Pure Chemicals. 2-, 3-, 4-Hydroxybenzoic acid, 2-, 3-methylbenzoic acid, 2-, 3-, 4methoxybenzoic acid, 4-ethoxybenzoic acid, and 3,4-dihydroxybenzoic (protocatechuic) acid were obtained from Sigma–Aldrich. All other chemicals were of analytical grade. Deionized water was obtained from Milli Q System (Millipore). Culture conditions. Phanerochaete chrysosporium (ATCC 34541) was grown from conidial inocula at 37 C in a stationary culture (20 mL medium in a 200-mL Erlenmeyer flask) under air. The medium (pH 4.5) used in this study with 28 mM D-glucose and 1.2 mM ammonium tartrate as the carbon and nitrogen sources, respectively, was previously described [12,16]. After 2-day preincubation, benzoic acid in acetonitrile (20 lL) was added to a final concentration of 1 mM. For the control culture, only acetonitrile (20 lL) was added. cDNA synthesis. After 24-h incubation in the absence or presence of benzoic acid, total RNA was isolated from P. chrysosporium using an RNeasy Plant Mini Kit (Qiagen). Reverse transcription (RT) was performed using oligo(dT)16 primer and Superscript II reverse transcriptase (Invitrogen) at 42 C for 50 min. The subsequent polymerase chain reaction (PCR) amplification was performed with the following primers: 5 0 -GAATTCAAAAATGTCTATGGCAGTAATTGAAGCA CTAACACAACTAGATTTGAAGTCGTG-3 0 and 5 0 -GCGGCCGC TCACAGGGTCCTCCTCCTGATA-3 0 . Primers were designed from the P. chrysosporium genomic sequence of ug.1.19.1 [http://genome. jgi-psf.org/whiterot1/whiterot1.home.html]. An EcoRI site (underlined) was introduced upstream of the yeast consensus sequence (indicated in bold) followed by the start codon and an NotI site (underlined) was introduced immediately downstream of the stop codon by PCR. Four silent mutations were added to the forward primer (double underlined). PCR amplification was performed using a DNA Thermal Cycler 2400 (Perkin-Elmer) as follows: initial denaturation at 95 C for 3 min, denaturing at 95 C for 30 s, annealing at 62 C for 30 s, and extension at 72 C for 2 min for 35 cycles. PCR products were separated by electrophoresis on 1.2% agarose gel, stained with ethidium bromide, and visualized using Molecular Imager FX (Bio-Rad). Obtained cDNA was designated PcCYP1f and its sequence was deposited in DDBJ under Accession No. AB219059. Cloning and sequencing. After purification using 1.2% agarose gel and a QIAquick gel extraction kit (Qiagen), PCR products were cloned into the pGEM-T Easy vector (Promega) and then transformed into Escherichia coli strain NovaBlue competent cells. Positive clones were selected by blue-white screening. Plasmids were isolated from positive clones using a QIAprep Spin Miniprep Kit (Qiagen) and sequenced with an automated DNA Sequencer (CEQ 8000; Beckman) using a DTCS Quick Start Kit (Beckman). The nucleotide and deduced amino acid sequences were analyzed using BLAST and FASTA search programs. Generation of recombinant P. pastoris containing PcCYP1f. A pPICZ A vector (Invitrogen) containing the methanol-inducible AOX1 promoter for control of gene expression and encoding resistance against zeocin was used to achieve intracellular expression of heterologous genes in the P. pastoris wild-type strain KM71H (Invitrogen). PcCYP1f cDNA digested by EcoRI and NotI was ligated into pPICZ A treated with the same endonucleases. E. coli strain NovaBlue was used for transformation and propagation of recombinant plasmids. Transformation of P. pastoris was achieved using EasySelect Pichia expression
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kit version G (Invitrogen). The presence of PcCYP1f in zeocin-resistant colonies was confirmed by direct PCR of the P. pastoris colonies. Pichia pastoris cultures and preparation of microsomes. Single colonies of P. pastoris were grown for 24 h at 30 C and 300 rpm in 200 mL BMGY (1% yeast extract, 2% peptone, 0.1 M potassium phosphate (pH 6.0), 1.34% yeast nitrogen base, 4 · 105% biotin, and 1% glycerol). Cells were harvested at 3000g for 5 min at room temperature and then inoculated in 50 mL of inducing medium (BMGY with 1% methanol instead of glycerol) in a 500-mL baffled flask. Cultures were grown for 24 h at 30 C and 300 rpm and the cells were pelleted at 3000g for 5 min at room temperature and then washed once in buffer A (50 mM potassium phosphate (pH 7.4), 1 mM EDTA, 20% glycerol, 1 mM DTT, and 0.25 mM phenylmethylsulfonyl fluoride) before being resuspended in buffer A to OD 130. The cells were broken by vortexing (10 · 30 s with intermediate cooling on ice) with an equal volume of acid-washed glass beads. The lysate was centrifuged at 11,000g for 10 min at 4 C to remove all debris and then the resulting supernatant was centrifuged at 130,000g for 1 h at 4 C to recover the microsomal pellet. Microsomes were resuspended in buffer A, stored at 80 C, and thawed on ice immediately before use. Catalytic activity of PcCYP1f. The P450 content in the microsomal fraction was determined by carbon monoxide-difference spectral analysis using an extinction coefficient of 91 mM1 cm1 [17]. PcCYP1f activity was measured using a microsomal fraction of P. pastoris containing recombinant PcCYP1f with exogenous addition of recombinant NADPH cytochrome P450 oxidoreductase from P. chrysosporium (PcCPR), which was expressed in E. coli and purified as described previously [18]. PcCPR activity was measured as NADPH-dependent cytochrome c reducing activity in reaction mixtures (1 mL) containing a PcCPR solution (100 lL), cytochrome c (10 lM), EDTA (0.5 mM), and NADPH (30 lM) in 50 mM potassium phosphate (pH 7.4). The reaction was initiated by adding NADPH. One unit of PcCPR is defined as the amount of enzyme that is able to catalyze the reduction of cytochrome c at an initial rate of one pmol per min. The rate of reduction of cytochrome c was determined spectrophotometrically at room temperature using 21.1 mM1 cm1 as the extinction coefficient for reduced minus oxidized cytochrome c at 550 nm [19,20]. No activity was observed when NADH was used instead of NADPH. Activity and substrate specificity of PcCYP1f were determined in reaction mixtures (500 lL) containing 100 lM PcCYP1f, 0.4 U PcCPR, 30 lg/lL L-a-dilauroyl phosphatidylcholine, 1 mM NADPH, 3 mM glucose-6-phosphate (G6P), 0.04 U glucose-6-phosphate dehydrogenase (G6PDH), and 5 lL of a substrate solution (0–600 mM in acetonitrile) in 50 mM potassium phosphate (pH 7.4). The reaction was initiated by addition of a substrate. After incubation of the reaction mixtures at 30 C for 15 min, 500 lL of 1 M HCl with 0.5 mM of cinnamic acid (internal standard) was added. Three types of control reactions, a zero-time control (terminated with 500 lL of 1 M HCl immediately after adding the substrate), no-NADPH control, and no-PcCYP1f control, were run parallel. The residual substrate and reaction products were analyzed by HPLC after filtration (0.45 lm) and by GCMS after extraction with 2 mL ethyl acetate (3·) and trimethylsilylation using N,O-bis(trimethylsilyl)trifluoroacetamide/pyridine (2:1, v/v). From the time course of the quantification data, the Michaelis constant (Km) and the maximum velocity (Vmax) were calculated. Real-time quantitative analysis of gene transcription. After 2-day preincubation of P. chrysosporium, benzoic acid was added. Total RNA was extracted from mycelia grown for an additional 12 or 24 h in the absence or presence of benzoic acid and applied (0.5 lg) to firststrand cDNA synthesis according to the methods described above. Real-time quantitative PCR was performed using LightCycler (Roche Molecular Biochemicals) with the following gene-specific primers 5 0 -ATGACAGTCTGCGGAAGGTT-3 0 and 5 0 -TTTTGCACTGCG TTCTTGTC-3 0 , generating a 149-bp amplicon. The PCR consisting of 2 lL of 1:32 diluted RT reaction product, 2 lL of LightCycler FastStart DNA Master SYBR Green I (Roche Molecular Biochemicals),
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2 lL of 5 lM forward primer, 2 lL of 5 lM reverse primer, 2.5 lL of 25 mM MgCl2, and 9.6 lL of sterile distilled water was initially incubated at 95 C for 10 min followed by amplification for up to 45 cycles of 95 C for 10 s, 55 C for 10 s, and 72 C for 10 s. Experimental samples were quantified by comparison with standards (b-globin) of known concentration (0.0003–30 ng) (Roche Molecular Biochemicals). The transcription level of the actin gene (Accession No. AB115328) was confirmed as a housekeeping target using the following primers; 5 0 -CCAAGGCTAACCGTGAGAAG-3 0 and 5 0 -CACCAGA GTCGAGCACGATA-3 0 , generating a 135-bp amplicon. Instrumentation. GCMS was performed at 70 eV on a JEOL AMII15 A equipped with a 30-m fused silica column (DB-5, J & W Scientific). The oven temperature was programmed from 80 to 320 C at 8 C/min with an injection temperature of 280 C. Products were identified by comparing their retention times on GC and HPLC and mass fragmentation patterns with authentic standards.
Results and discussion Structure of the PcCYP1f gene from P. chrysosporium Full-length PcCYP1f cDNA encoding 536 amino acid residues with a calculated molecular weight of 60.0 kDa was cloned (Fig. 1). PcCYP1f contains motifs characteristic of P450 [21], such as distal helices and a heme-binding domain (FSFGPRSCVG), at residues 421–430 in the C terminal region (Fig. 1). Using the cDNA sequence, the genomic PcCYP1f was found to possess five introns and six exons within a span of 2062 bp. All intron–exon boundaries conformed to the canonical GT-AG rule. The length of introns ranged from 50 to 231 bp. The sizes of the exons ranged widely from 72 to 424 bp for the sixth and third exons, respectively. The predicted amino acid sequence of PcCYP1f was highly homologous with that of P450s of the CYP53 family, with 52% identity to CYP53B1 and 49% to CYP53A1 and CYP53A3 (Fig. 1) [13,15,22]. Aspergillus niger CYP53A1 (P450-bphA) was the first enzyme that was shown to catalyze the hydroxylation of benzoic acid at the 4-position, which is an essential step for benzoate catabolism in this fungus [22]. This subfamily was later joined by CYP53B1 (P450rm) of the basidiomycete yeast Rhodotorula minuta and by an uncharacterized gene of Aspergillus parasiticus (CYP53A2) [13,15,21,22]. It was suggested that PcCYP1f belongs to a novel subfamily of the CYP53 family. Conservation between PcCYP1f and other members of the CYP53 family is particularly high in the distal helix region and substrate recognition sites (SRSs) 1, 4, 5, and 6 (Fig. 1). SRSs conserved among members of the CYP53 family are thought to be required for interaction with a common substrate, benzoic acid. Functional expression of PcCYP1f in P. pastoris Several heme-containing proteins, such as nitric oxide synthase [23], horseradish peroxidase [24], and manga-
nese peroxidase [25], have been successfully applied to express in the methylotrophic yeast P. pastoris. Thus, P. pastoris was explored as an expression system for PcCYP1f. SDS–PAGE analysis indicated a new protein band in the microsomal fraction prepared from PcCYP1f-transformed P. pastoris upon induction (Fig. 2A, inset). Many eukaryotic P450s are localized in the microsomal fraction of the cell [26]. The molecular mass of 60 kDa of the newly appeared band was consistent with the molecular weight predicted from the deduced amino acid sequence (60.0 kDa). A typical P450 CO-difference spectrum with an absorption maximum at 448 nm and no maximum at 420 nm was observed in the microsomal fraction from PcCYP1f-transformed P. pastoris with addition of inducer but not without addition (Fig. 2A), clearly indicating the formation of functionally active P450 molecular species [17]. PcCYP1f generated a type I substrate binding spectrum with benzoic acid (Fig. 2B), indicating a shift from low to high spin state upon substrate binding [27]. Finally, we determined the catalytic activity in the microsomal fraction from induced cells. HPLC and GCMS analyses clearly indicated the formation of 4hydroxybenzoic acid from benzoic acid in the presence of NADPH and PcCPR: 4-hydroxybenzoic acid (diTMS) m/z; 282 (M+ 12%), 267 (59), 223 (66), 193 (53), and 73 (100). No other products were seen. P450-catalyzed formation of 4-hydroxybenzoate was linear with the enzyme concentration and incubation time under the present conditions. No conversion took place using the P. pastoris microsomal fraction prepared from non-induced cells (data not shown). Thus, PcCYP1f catalyzes the hydroxylation of benzoic acid at the 4-position as reported for CYP53 P450s [13–15]. Steady-state kinetic analysis indicated a Km of 185 lM and Vmax of 0.013 lmol/min/lmol for PcCYP1f-catalyzed hydroxylation of benzoic acid. These kinetic parameters were similar to those for CYP53A1 [28], indicating that the catalytic efficiency of PcCYP1f is as good as benzoate4-hydroxylase. Substrate specificity of PcCYP1f PcCYP1f also generated a typical type I substrate binding spectrum with several single substituted benzoic acids, such as 2- and 3-methylbenzoic acid, and 3hydroxybenzoic acid. Catalytic activity of PcCYP1f against these compounds was examined, indicating decrease of 2- and 3-methylbenzoic acid, and 3hydroxybenzoic acid during the course of the reactions. 3,4-Dihydroxybenzoic acid (protocatechuic acid) was identified as a reaction product of 3-hydroxybezoic acid by HPLC and GCMS: protocatechuic acid (triTMS) m/z; 370 (M+ 49%), 354 (23), 311 (32), 281 (13), 223 (6), 193 (81), and 73 (100). On the contrary, 2-, 3-, and 4-methoxy benzoic acid, 2- and 4-hydroxy-
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Fig. 1. Amino acid sequences of PcCYP1f and CYP53 P450s. PcCYP is from P. chrysosporium PcCYP1f (Accession No. AAQ84022), CYP53A1 from Aspergillus nigar (P17549), CYP53A3 from Aspergillus nidulans (AY048583), and CYP53B1 from Rhodotorula minuta (BAA09832). Single lines show substrate recognition sites (SRSs) and the broken line indicates the distal helix region. The heme-binding domain is boxed.
benzoic acid, and 4-ethoxybenzoic acid were not hydroxylated by PcCYP1f. CYP53A1 reportedly catalyzes the hydroxylation of 2-hydroxybenzoic acid but not 3-hydroxybenzoic acid [28], suggesting that
PcCYP1f has a slightly different substrate specificity than other members in the CYP53 family. In our previous report, upon exogenous addition of benzoic acid to P. chrysosporium, 2- and 4-hydroxybenzoic acid,
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1
and 4-hydroxy-3-methoxybezoic (vanillic) acid were identified as metabolites [12]. In the present study, PcCYP1f was shown to catalyze the hydroxylation of benzoic acid to 4-hydroxybenzoic acid but not to 2-hydroxybenzoic acid, strongly suggesting the existence of another P450 responsible for 2-hydroxybenzoic acid formation. Furthermore, 3-hydroxybenzoic acid was shown to be a substrate for PcCYP1f but 4-hydroxybenzoic acid was not. Other fungal P450(s) are thought to be involved in the formation of protocatechuic acid, a possible precursor of vanillic acid production (Fig. 3). In addition, protocatechuic acid is known to be a key intermediate in the b-ketoadipate pathway during the degradation of aromatic compounds [29,30]; thus, PcCYP1f is thought to play a crucial role in benzoate metabolism.
2
75 kDa
Absorbance
448
50 kDa
35 kDa
400
450 Wavelength (nm)
500
Absorbance
392
Regulatory effect of benzoate on PcCYP1f expression in P. chrysosporium Real-time quantitative PCR analysis was utilized to examine the inducibility of PcCYP1f by benzoic acid. Total RNA was prepared after 12- and 24-h incubation of P. chrysosporium either in the presence or absence of benzoic acid. The amplicons of b-actin from samples treated in an identical manner served as a quantitative control, because expression was not affected by different doses of benzoic acid. As shown in Fig. 4, the number of PcCYP1f transcripts was 1.6- and 2.4-fold higher after 12- and 24-h incubation with benzoic acid, respectively, compared to the control. This observation strongly suggests that the expression of PcCYP1f is regulated by benzoic acid at the transcription level.
427
360
400 440 Wavelength (nm)
480
Fig. 2. Difference spectra of recombinant PcCYP1f. (A) Difference spectrum upon addition of carbon monoxide to reduced microsomal PcCYP1f. (B) Difference spectrum upon addition of benzoic acid. Inset shows the SDS–PAGE results of the P. pastoris microsome without (lane 1) or with (lane 2) the addition of an inducer of PcCYP1f expression. The arrow indicates a newly appeared protein corresponding to PcCYP1f.
COOH
COOH
PcCYP1f OH PcCYP?
PcCYP?
COOH
COOH
COOH
PcCYP1f OH
OH OH
OCH 3 OH
Fig. 3. Involvement of PcCYP1f in benzoate metabolism. Involvement of other fungal P450(s) is also suggested.
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Fold induction
2.5 2.0 1.5 1.0 0.5 0 12
24 Incubation time (hour)
Fig. 4. Real-time RT-PCR study of PcCYP1f expression in response to exogenous addition of benzoic acid. A 149-bp fragment of PcCYP1f cDNA was amplified from reverse-transcribed RNA samples of P. chrysosporium cells with or without addition of benzoic acid. The PcCYP1f-data were normalized by comparison with amplification of 135-bp b-actin cDNA. Open and closed bars indicate relative expression amounts of PcCYP1f in the absence and presence of exogenous benzoic acid, respectively.
In the present study, we reported the isolation and characterization of a cDNA clone of PcCYP1f from the lignin-degrading basidiomycete P. chrysosporium, indicating that it belongs to a new subfamily of the CYP53 family. Furthermore, the expression of enzymatically active P. chrysosporium PcCYP1f was successfully achieved using the P. pastoris heterologous expression system, demonstrating that PcCYP1f is capable of hydroxylating 3-hydroxybenzoic acid as well as benzoic acid at the 4-position. This is the first report showing the expression of P450 from P. chrysosporium in an enzymatically active form. Application of the expression system with other P. chrysosporium P450s is now under way.
Acknowledgments This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (to H.W). The authors thank Professors M. Okamoto and T. Hanai for their help in real-time PCR quantitative analysis.
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[25] L. Gu, C. Lajoie, C. Kelly, Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris, Biotechnol. Prog. 19 (2003) 1403–1409. [26] T. Omura, Y. Ishimura, Y. Fujii-Kuriyama (Eds.), Cytochrome P-450, second ed., Kodansha, Tokyo, Japan, 1993. [27] R. Raag, T.L. Poulos, Crystal structures of cytochrome P-450CAM complexed with camphane, thiocamphor, and adamantane: factors controlling P-450 substrate hydroxylation, Biochemistry 30 (1991) 2674–2684. [28] B.W. Faber, R.F. van Gorcom, J.A. Duine, Purification and characterization of benzoate-para-hydroxylase, a cytochrome P450 (CYP53A1), from Aspergillus niger, Arch. Biochem. Biophys. 394 (2001) 245–254. [29] W.J. Middelhoven, Catabolism of benzene compounds by ascomycetous and basidiomycetous yeasts and yeast-like fungi: a literature review and an experimental approach, Antonie Van Leeuwenhoek 63 (1993) 125–144. [30] M. Wojtas-Wasilewska, J. Trojanowski, J. Luterek, Aromatic ring cleavage of protocatechuic acid by the white-rot fungus Pleurotus ostreatus, Acta Biochim. Pol. 30 (1983) 291–302.
Molecular characterization of cytochrome P450 catalyzing hydroxylation of benzoates from the white-rot fungus Phanerochaete chrysosporium Fumiko Matsuzaki, Hiroyuki Wariishi * Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan Received 1 July 2005 Available online 18 July 2005
Abstract We cloned full-length cDNA (PcCYP1f) encoding one of the cytochrome P450s in the lignin-degrading basidiomycete Phanerochaete chrysosporium, which showed high homology to P450s in the CYP53 family. PcCYP1f was expressed as an active microsomal protein using the methylotrophic yeast Pichia pastoris expression system. Using the microsomal fraction containing PcCYP1f, a typical P450 CO-difference spectrum was obtained with absorption maximum at 448 nm. Recombinant PcCYP1f catalyzed the hydroxylation of benzoic acid into 4-hydroxybenzoic acid in the presence of NADPH and P. chrysosporium cytochrome P450 oxidoreductase. In contrast to other CYP53 P450s, this enzyme was shown to catalyze the hydroxylation of 3-hydroxybenzoate into 3,4-dihydroxybenzoate. Furthermore, 2- and 3-methylbenzoate were also shown to be substrates of PcCYP1f. This is the first report showing the expression of a functionally active Phanerochaete P450. Finally, real-time quantitative PCR analysis revealed that PcCYP1f is induced at a transcriptional level by exogenous addition of benzoic acid. 2005 Elsevier Inc. All rights reserved. Keywords: Basidiomycete; Benzoate hydroxylase; Carbon monoxide-difference spectrum; Cytochrome P450; Heterologous expression; Kinetic analysis; Phanerochaete chrysosporium; Pichia pastoris; Real-time PCR; Substrate binding spectrum
White-rot fungi are capable of degrading a wide variety of recalcitrant aromatic compounds, including polymeric lignin and environmentally persistent pollutants. Extracellular ligninolytic enzymes, such as lignin and manganese peroxidases, involved in the metabolism of aromatic compounds have been extensively studied [1,2]. Intracellularly, cytochrome P450 (P450)-mediated oxygenation reactions are known to play an important role during fungal metabolism of recalcitrant xenobiotic compounds [3–10]. Phanerochaete chrysosporium is the most extensively studied white-rot fungus with regard to ligninolysis and xenobiotic metabolism. The gene diversity of fungal P450 was recently suggested via the whole sequence of the P. chrysosporium genome, where *
Corresponding author. Fax: +81 92 642 2992. E-mail address: [email protected] (H. Wariishi).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.013
as many as 148 P450 genes have been found [11]. Recently, we utilized a series of well-characterized P450 substrates as metabolic substrates for P. chrysosporium, pointing out not only the gene diversity but also the functional diversity of fungal P450 molecular species. Among the substrates examined, benzoate was found to be converted by Phanerochaete P450(s) [12]. In the present study, we cloned PcCYP1f, one of the Phanerochaete P450 genes, and showed high homology to the P450s in the CYP53 family (benzoate-4-hydroxylase), which are known to catalyze the hydroxylation of benzoic acid at the 4-position [13–15]. PcCYP1f was expressed in the heterologous expression system using Pichia pastoris. In the CO-difference spectrum, recombinant PcCYP1f exhibited absorption maximum at 448 nm with no contamination of absorption at 420 nm. It was also shown to catalyze the hydroxylation of benzoic acid
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into 4-hydroxybenzoic acid in the presence of NADPH and P. chrysosporium cytochrome P450 reductase (PcCPR). Finally, real-time quantitative PCR analysis revealed that PcCYP1f was induced at the transcriptional level by exogenous addition of benzoic acid.
Materials and methods Chemicals. Benzoic acid was purchased from Wako Pure Chemicals. 2-, 3-, 4-Hydroxybenzoic acid, 2-, 3-methylbenzoic acid, 2-, 3-, 4methoxybenzoic acid, 4-ethoxybenzoic acid, and 3,4-dihydroxybenzoic (protocatechuic) acid were obtained from Sigma–Aldrich. All other chemicals were of analytical grade. Deionized water was obtained from Milli Q System (Millipore). Culture conditions. Phanerochaete chrysosporium (ATCC 34541) was grown from conidial inocula at 37 C in a stationary culture (20 mL medium in a 200-mL Erlenmeyer flask) under air. The medium (pH 4.5) used in this study with 28 mM D-glucose and 1.2 mM ammonium tartrate as the carbon and nitrogen sources, respectively, was previously described [12,16]. After 2-day preincubation, benzoic acid in acetonitrile (20 lL) was added to a final concentration of 1 mM. For the control culture, only acetonitrile (20 lL) was added. cDNA synthesis. After 24-h incubation in the absence or presence of benzoic acid, total RNA was isolated from P. chrysosporium using an RNeasy Plant Mini Kit (Qiagen). Reverse transcription (RT) was performed using oligo(dT)16 primer and Superscript II reverse transcriptase (Invitrogen) at 42 C for 50 min. The subsequent polymerase chain reaction (PCR) amplification was performed with the following primers: 5 0 -GAATTCAAAAATGTCTATGGCAGTAATTGAAGCA CTAACACAACTAGATTTGAAGTCGTG-3 0 and 5 0 -GCGGCCGC TCACAGGGTCCTCCTCCTGATA-3 0 . Primers were designed from the P. chrysosporium genomic sequence of ug.1.19.1 [http://genome. jgi-psf.org/whiterot1/whiterot1.home.html]. An EcoRI site (underlined) was introduced upstream of the yeast consensus sequence (indicated in bold) followed by the start codon and an NotI site (underlined) was introduced immediately downstream of the stop codon by PCR. Four silent mutations were added to the forward primer (double underlined). PCR amplification was performed using a DNA Thermal Cycler 2400 (Perkin-Elmer) as follows: initial denaturation at 95 C for 3 min, denaturing at 95 C for 30 s, annealing at 62 C for 30 s, and extension at 72 C for 2 min for 35 cycles. PCR products were separated by electrophoresis on 1.2% agarose gel, stained with ethidium bromide, and visualized using Molecular Imager FX (Bio-Rad). Obtained cDNA was designated PcCYP1f and its sequence was deposited in DDBJ under Accession No. AB219059. Cloning and sequencing. After purification using 1.2% agarose gel and a QIAquick gel extraction kit (Qiagen), PCR products were cloned into the pGEM-T Easy vector (Promega) and then transformed into Escherichia coli strain NovaBlue competent cells. Positive clones were selected by blue-white screening. Plasmids were isolated from positive clones using a QIAprep Spin Miniprep Kit (Qiagen) and sequenced with an automated DNA Sequencer (CEQ 8000; Beckman) using a DTCS Quick Start Kit (Beckman). The nucleotide and deduced amino acid sequences were analyzed using BLAST and FASTA search programs. Generation of recombinant P. pastoris containing PcCYP1f. A pPICZ A vector (Invitrogen) containing the methanol-inducible AOX1 promoter for control of gene expression and encoding resistance against zeocin was used to achieve intracellular expression of heterologous genes in the P. pastoris wild-type strain KM71H (Invitrogen). PcCYP1f cDNA digested by EcoRI and NotI was ligated into pPICZ A treated with the same endonucleases. E. coli strain NovaBlue was used for transformation and propagation of recombinant plasmids. Transformation of P. pastoris was achieved using EasySelect Pichia expression
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kit version G (Invitrogen). The presence of PcCYP1f in zeocin-resistant colonies was confirmed by direct PCR of the P. pastoris colonies. Pichia pastoris cultures and preparation of microsomes. Single colonies of P. pastoris were grown for 24 h at 30 C and 300 rpm in 200 mL BMGY (1% yeast extract, 2% peptone, 0.1 M potassium phosphate (pH 6.0), 1.34% yeast nitrogen base, 4 · 105% biotin, and 1% glycerol). Cells were harvested at 3000g for 5 min at room temperature and then inoculated in 50 mL of inducing medium (BMGY with 1% methanol instead of glycerol) in a 500-mL baffled flask. Cultures were grown for 24 h at 30 C and 300 rpm and the cells were pelleted at 3000g for 5 min at room temperature and then washed once in buffer A (50 mM potassium phosphate (pH 7.4), 1 mM EDTA, 20% glycerol, 1 mM DTT, and 0.25 mM phenylmethylsulfonyl fluoride) before being resuspended in buffer A to OD 130. The cells were broken by vortexing (10 · 30 s with intermediate cooling on ice) with an equal volume of acid-washed glass beads. The lysate was centrifuged at 11,000g for 10 min at 4 C to remove all debris and then the resulting supernatant was centrifuged at 130,000g for 1 h at 4 C to recover the microsomal pellet. Microsomes were resuspended in buffer A, stored at 80 C, and thawed on ice immediately before use. Catalytic activity of PcCYP1f. The P450 content in the microsomal fraction was determined by carbon monoxide-difference spectral analysis using an extinction coefficient of 91 mM1 cm1 [17]. PcCYP1f activity was measured using a microsomal fraction of P. pastoris containing recombinant PcCYP1f with exogenous addition of recombinant NADPH cytochrome P450 oxidoreductase from P. chrysosporium (PcCPR), which was expressed in E. coli and purified as described previously [18]. PcCPR activity was measured as NADPH-dependent cytochrome c reducing activity in reaction mixtures (1 mL) containing a PcCPR solution (100 lL), cytochrome c (10 lM), EDTA (0.5 mM), and NADPH (30 lM) in 50 mM potassium phosphate (pH 7.4). The reaction was initiated by adding NADPH. One unit of PcCPR is defined as the amount of enzyme that is able to catalyze the reduction of cytochrome c at an initial rate of one pmol per min. The rate of reduction of cytochrome c was determined spectrophotometrically at room temperature using 21.1 mM1 cm1 as the extinction coefficient for reduced minus oxidized cytochrome c at 550 nm [19,20]. No activity was observed when NADH was used instead of NADPH. Activity and substrate specificity of PcCYP1f were determined in reaction mixtures (500 lL) containing 100 lM PcCYP1f, 0.4 U PcCPR, 30 lg/lL L-a-dilauroyl phosphatidylcholine, 1 mM NADPH, 3 mM glucose-6-phosphate (G6P), 0.04 U glucose-6-phosphate dehydrogenase (G6PDH), and 5 lL of a substrate solution (0–600 mM in acetonitrile) in 50 mM potassium phosphate (pH 7.4). The reaction was initiated by addition of a substrate. After incubation of the reaction mixtures at 30 C for 15 min, 500 lL of 1 M HCl with 0.5 mM of cinnamic acid (internal standard) was added. Three types of control reactions, a zero-time control (terminated with 500 lL of 1 M HCl immediately after adding the substrate), no-NADPH control, and no-PcCYP1f control, were run parallel. The residual substrate and reaction products were analyzed by HPLC after filtration (0.45 lm) and by GCMS after extraction with 2 mL ethyl acetate (3·) and trimethylsilylation using N,O-bis(trimethylsilyl)trifluoroacetamide/pyridine (2:1, v/v). From the time course of the quantification data, the Michaelis constant (Km) and the maximum velocity (Vmax) were calculated. Real-time quantitative analysis of gene transcription. After 2-day preincubation of P. chrysosporium, benzoic acid was added. Total RNA was extracted from mycelia grown for an additional 12 or 24 h in the absence or presence of benzoic acid and applied (0.5 lg) to firststrand cDNA synthesis according to the methods described above. Real-time quantitative PCR was performed using LightCycler (Roche Molecular Biochemicals) with the following gene-specific primers 5 0 -ATGACAGTCTGCGGAAGGTT-3 0 and 5 0 -TTTTGCACTGCG TTCTTGTC-3 0 , generating a 149-bp amplicon. The PCR consisting of 2 lL of 1:32 diluted RT reaction product, 2 lL of LightCycler FastStart DNA Master SYBR Green I (Roche Molecular Biochemicals),
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2 lL of 5 lM forward primer, 2 lL of 5 lM reverse primer, 2.5 lL of 25 mM MgCl2, and 9.6 lL of sterile distilled water was initially incubated at 95 C for 10 min followed by amplification for up to 45 cycles of 95 C for 10 s, 55 C for 10 s, and 72 C for 10 s. Experimental samples were quantified by comparison with standards (b-globin) of known concentration (0.0003–30 ng) (Roche Molecular Biochemicals). The transcription level of the actin gene (Accession No. AB115328) was confirmed as a housekeeping target using the following primers; 5 0 -CCAAGGCTAACCGTGAGAAG-3 0 and 5 0 -CACCAGA GTCGAGCACGATA-3 0 , generating a 135-bp amplicon. Instrumentation. GCMS was performed at 70 eV on a JEOL AMII15 A equipped with a 30-m fused silica column (DB-5, J & W Scientific). The oven temperature was programmed from 80 to 320 C at 8 C/min with an injection temperature of 280 C. Products were identified by comparing their retention times on GC and HPLC and mass fragmentation patterns with authentic standards.
Results and discussion Structure of the PcCYP1f gene from P. chrysosporium Full-length PcCYP1f cDNA encoding 536 amino acid residues with a calculated molecular weight of 60.0 kDa was cloned (Fig. 1). PcCYP1f contains motifs characteristic of P450 [21], such as distal helices and a heme-binding domain (FSFGPRSCVG), at residues 421–430 in the C terminal region (Fig. 1). Using the cDNA sequence, the genomic PcCYP1f was found to possess five introns and six exons within a span of 2062 bp. All intron–exon boundaries conformed to the canonical GT-AG rule. The length of introns ranged from 50 to 231 bp. The sizes of the exons ranged widely from 72 to 424 bp for the sixth and third exons, respectively. The predicted amino acid sequence of PcCYP1f was highly homologous with that of P450s of the CYP53 family, with 52% identity to CYP53B1 and 49% to CYP53A1 and CYP53A3 (Fig. 1) [13,15,22]. Aspergillus niger CYP53A1 (P450-bphA) was the first enzyme that was shown to catalyze the hydroxylation of benzoic acid at the 4-position, which is an essential step for benzoate catabolism in this fungus [22]. This subfamily was later joined by CYP53B1 (P450rm) of the basidiomycete yeast Rhodotorula minuta and by an uncharacterized gene of Aspergillus parasiticus (CYP53A2) [13,15,21,22]. It was suggested that PcCYP1f belongs to a novel subfamily of the CYP53 family. Conservation between PcCYP1f and other members of the CYP53 family is particularly high in the distal helix region and substrate recognition sites (SRSs) 1, 4, 5, and 6 (Fig. 1). SRSs conserved among members of the CYP53 family are thought to be required for interaction with a common substrate, benzoic acid. Functional expression of PcCYP1f in P. pastoris Several heme-containing proteins, such as nitric oxide synthase [23], horseradish peroxidase [24], and manga-
nese peroxidase [25], have been successfully applied to express in the methylotrophic yeast P. pastoris. Thus, P. pastoris was explored as an expression system for PcCYP1f. SDS–PAGE analysis indicated a new protein band in the microsomal fraction prepared from PcCYP1f-transformed P. pastoris upon induction (Fig. 2A, inset). Many eukaryotic P450s are localized in the microsomal fraction of the cell [26]. The molecular mass of 60 kDa of the newly appeared band was consistent with the molecular weight predicted from the deduced amino acid sequence (60.0 kDa). A typical P450 CO-difference spectrum with an absorption maximum at 448 nm and no maximum at 420 nm was observed in the microsomal fraction from PcCYP1f-transformed P. pastoris with addition of inducer but not without addition (Fig. 2A), clearly indicating the formation of functionally active P450 molecular species [17]. PcCYP1f generated a type I substrate binding spectrum with benzoic acid (Fig. 2B), indicating a shift from low to high spin state upon substrate binding [27]. Finally, we determined the catalytic activity in the microsomal fraction from induced cells. HPLC and GCMS analyses clearly indicated the formation of 4hydroxybenzoic acid from benzoic acid in the presence of NADPH and PcCPR: 4-hydroxybenzoic acid (diTMS) m/z; 282 (M+ 12%), 267 (59), 223 (66), 193 (53), and 73 (100). No other products were seen. P450-catalyzed formation of 4-hydroxybenzoate was linear with the enzyme concentration and incubation time under the present conditions. No conversion took place using the P. pastoris microsomal fraction prepared from non-induced cells (data not shown). Thus, PcCYP1f catalyzes the hydroxylation of benzoic acid at the 4-position as reported for CYP53 P450s [13–15]. Steady-state kinetic analysis indicated a Km of 185 lM and Vmax of 0.013 lmol/min/lmol for PcCYP1f-catalyzed hydroxylation of benzoic acid. These kinetic parameters were similar to those for CYP53A1 [28], indicating that the catalytic efficiency of PcCYP1f is as good as benzoate4-hydroxylase. Substrate specificity of PcCYP1f PcCYP1f also generated a typical type I substrate binding spectrum with several single substituted benzoic acids, such as 2- and 3-methylbenzoic acid, and 3hydroxybenzoic acid. Catalytic activity of PcCYP1f against these compounds was examined, indicating decrease of 2- and 3-methylbenzoic acid, and 3hydroxybenzoic acid during the course of the reactions. 3,4-Dihydroxybenzoic acid (protocatechuic acid) was identified as a reaction product of 3-hydroxybezoic acid by HPLC and GCMS: protocatechuic acid (triTMS) m/z; 370 (M+ 49%), 354 (23), 311 (32), 281 (13), 223 (6), 193 (81), and 73 (100). On the contrary, 2-, 3-, and 4-methoxy benzoic acid, 2- and 4-hydroxy-
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Fig. 1. Amino acid sequences of PcCYP1f and CYP53 P450s. PcCYP is from P. chrysosporium PcCYP1f (Accession No. AAQ84022), CYP53A1 from Aspergillus nigar (P17549), CYP53A3 from Aspergillus nidulans (AY048583), and CYP53B1 from Rhodotorula minuta (BAA09832). Single lines show substrate recognition sites (SRSs) and the broken line indicates the distal helix region. The heme-binding domain is boxed.
benzoic acid, and 4-ethoxybenzoic acid were not hydroxylated by PcCYP1f. CYP53A1 reportedly catalyzes the hydroxylation of 2-hydroxybenzoic acid but not 3-hydroxybenzoic acid [28], suggesting that
PcCYP1f has a slightly different substrate specificity than other members in the CYP53 family. In our previous report, upon exogenous addition of benzoic acid to P. chrysosporium, 2- and 4-hydroxybenzoic acid,
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1
and 4-hydroxy-3-methoxybezoic (vanillic) acid were identified as metabolites [12]. In the present study, PcCYP1f was shown to catalyze the hydroxylation of benzoic acid to 4-hydroxybenzoic acid but not to 2-hydroxybenzoic acid, strongly suggesting the existence of another P450 responsible for 2-hydroxybenzoic acid formation. Furthermore, 3-hydroxybenzoic acid was shown to be a substrate for PcCYP1f but 4-hydroxybenzoic acid was not. Other fungal P450(s) are thought to be involved in the formation of protocatechuic acid, a possible precursor of vanillic acid production (Fig. 3). In addition, protocatechuic acid is known to be a key intermediate in the b-ketoadipate pathway during the degradation of aromatic compounds [29,30]; thus, PcCYP1f is thought to play a crucial role in benzoate metabolism.
2
75 kDa
Absorbance
448
50 kDa
35 kDa
400
450 Wavelength (nm)
500
Absorbance
392
Regulatory effect of benzoate on PcCYP1f expression in P. chrysosporium Real-time quantitative PCR analysis was utilized to examine the inducibility of PcCYP1f by benzoic acid. Total RNA was prepared after 12- and 24-h incubation of P. chrysosporium either in the presence or absence of benzoic acid. The amplicons of b-actin from samples treated in an identical manner served as a quantitative control, because expression was not affected by different doses of benzoic acid. As shown in Fig. 4, the number of PcCYP1f transcripts was 1.6- and 2.4-fold higher after 12- and 24-h incubation with benzoic acid, respectively, compared to the control. This observation strongly suggests that the expression of PcCYP1f is regulated by benzoic acid at the transcription level.
427
360
400 440 Wavelength (nm)
480
Fig. 2. Difference spectra of recombinant PcCYP1f. (A) Difference spectrum upon addition of carbon monoxide to reduced microsomal PcCYP1f. (B) Difference spectrum upon addition of benzoic acid. Inset shows the SDS–PAGE results of the P. pastoris microsome without (lane 1) or with (lane 2) the addition of an inducer of PcCYP1f expression. The arrow indicates a newly appeared protein corresponding to PcCYP1f.
COOH
COOH
PcCYP1f OH PcCYP?
PcCYP?
COOH
COOH
COOH
PcCYP1f OH
OH OH
OCH 3 OH
Fig. 3. Involvement of PcCYP1f in benzoate metabolism. Involvement of other fungal P450(s) is also suggested.
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Fold induction
2.5 2.0 1.5 1.0 0.5 0 12
24 Incubation time (hour)
Fig. 4. Real-time RT-PCR study of PcCYP1f expression in response to exogenous addition of benzoic acid. A 149-bp fragment of PcCYP1f cDNA was amplified from reverse-transcribed RNA samples of P. chrysosporium cells with or without addition of benzoic acid. The PcCYP1f-data were normalized by comparison with amplification of 135-bp b-actin cDNA. Open and closed bars indicate relative expression amounts of PcCYP1f in the absence and presence of exogenous benzoic acid, respectively.
In the present study, we reported the isolation and characterization of a cDNA clone of PcCYP1f from the lignin-degrading basidiomycete P. chrysosporium, indicating that it belongs to a new subfamily of the CYP53 family. Furthermore, the expression of enzymatically active P. chrysosporium PcCYP1f was successfully achieved using the P. pastoris heterologous expression system, demonstrating that PcCYP1f is capable of hydroxylating 3-hydroxybenzoic acid as well as benzoic acid at the 4-position. This is the first report showing the expression of P450 from P. chrysosporium in an enzymatically active form. Application of the expression system with other P. chrysosporium P450s is now under way.
Acknowledgments This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (to H.W). The authors thank Professors M. Okamoto and T. Hanai for their help in real-time PCR quantitative analysis.
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