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Cloning, sequencing, and expression in Escherichia coli of a cytochrome P450 gene from Cunninghamella elegans
FEMS Microbiology Letters 188 (2000) 55^61
www.fems-microbiology.org
Cloning, sequencing, and expression in Escherichia coli of a cytochrome P450 gene from Cunninghamella elegans Rong-Fu Wang, Wei-Wen Cao, Ashraf A. Khan, Carl E. Cerniglia * Division of Microbiology, National Center for Toxicological Research, FDA, Je¡erson, AR 72079-9502, USA Received 22 November 1999; received in revised form 8 May 2000; accepted 8 May 2000
Abstract A polyclonal antibody against microsomes of a fungus, Cunninghamella elegans, was used to screen a C. elegans cDNA library. A cDNA clone, containing an open reading frame (ORF) encoding a protein of 389 amino acids (aa), was obtained. GenBank comparison (BLAST) showed that the protein was closely related to P450 because a heme-binding region, which is highly conserved in all P450 sequences, was found in the ORF protein. Using an oligo probe designed from this C. elegans heme-binding region to rescreen the cDNA library, we obtained three new clones. Sequence comparison showed that the three clones, with different length cDNA inserts, were from the same mRNA of the C. elegans P450 gene. One clone had the full C. elegans P450 gene, encoding 473 aa with a molecular mass of 54 958.60, whereas the 389 was a part of the 473 aa without the N-terminal. The entire C. elegans P450 gene was successfully subcloned and overexpressed in a plasmid^Escherichia coli system (pQE30). Immunostaining with three antibodies (CYP1A1, CYP2E1, and CYP3A1) against mammalian P450 enzymes and benzidine staining for hemoproteins showed positive results for the recombinant protein expressed in E. coli. A phylogenetic tree was constructed by comparison of other fungal P450s to the C. elegans sequence. The C. elegans P450 clustered close to the cyp51 family and was named cyp509A1 by the International Committee on the Nomenclature for Cytochrome P450 Enzymes. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : P450; Cloning; Expression ; Cunninghamella elegans
1. Introduction The ¢lamentous fungus, Cunninghamella elegans, is a zygomycete, which has been used as a microbial model of mammalian metabolism and for the biodegradation of priority pollutants [1^4]. C. elegans ATCC 36112 metabolizes or biotransforms numerous structurally diverse compounds, such as polycyclic aromatic hydrocarbons (PAHs), crude oil components, drugs, and N-,S-,O-heterocyclic aromatic compounds, by phase I and phase II enzymes [5]. The phase I reactions catalyzed by cytochrome P450 in C. elegans include aliphatic and aromatic hydroxylation, N- and O-dealkylation and N- and S-oxidation [5^ 11]. The involvement of cytochrome P450 enzymes in the metabolism of PAHs and pharmaceutical compounds by C. elegans is based on carbon monoxide di¡erence spectra (absorption at 450 nm), enzyme activities, cytochrome P450 inhibitors, 18 O2 incorporation experiments,
and spectra of metabolites that are similar to those formed by mammalian cytochrome P450 enzyme systems [2,8,9]. Little is known about the molecular genetics of C. elegans, except for studies of the enolase and the 6-phosphogluconate dehydrogenase conducted in this laboratory [12,13]. The use of molecular biology techniques to characterize fungal cytochrome P450 enzyme systems has greatly enhanced our understanding of fungal biotransformation of xenobiotics, especially since it is di¤cult to purify fungal microsomal P450 proteins using conventional biochemical techniques [14]. Since many of the phase I and phase II enzymes are localized in the microsomal fraction of the cell [5], a polyclonal antibody against C. elegans microsomes was used to screen a C. elegans cDNA library in this study. The isolation and characterization of three cDNA clones encoding a C. elegans cytochrome P450 is reported in this article.
* Corresponding author. Tel. : +1 (870) 543-7341; Fax: +1 (870) 543-7307; E-mail: [email protected] 0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 2 1 0 - X
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2. Materials and methods 2.1. Mycelia, microsomes, cDNA library and rabbit antiserum preparations C. elegans ATCC 36112 was grown in Sabouraud dextrose broth (Difco Laboratories, Detroit, MI) at room temperature without shaking. After 5 days of incubation, the mycelium was harvested by ¢ltration and washed with deionized water. Freshly prepared C. elegans mycelium was frozen in liquid nitrogen and sent on dry ice to Clontech Laboratories, Inc. (Palo Alto, CA) for custom mRNA preparation and custom cDNA library construction. Microsomes isolated from C. elegans mycelia [5] were sent to Alpha Diagnostic (San Antonio, TX) for custom antiserum preparation from rabbit (the antibody titer was 104 ). 2.2. Screening the cDNA library, ampli¢cation and DNA sequence analysis Rabbit antiserum raised to C. elegans microsomes was used to screen the unampli¢ed C. elegans cDNA library. The cDNA library (5U103 /90-mm plate) was mixed with Escherichia coli Y1090, which had been cultured in LB broth [15]^0.2% maltose^10 mM MgSO4 . After 20 min adsorption, 3 ml LB-soft top agarose (0.6%) at 55³C was added and immediately poured onto an LB agar plate. The plate was incubated at 42³C for 3.5 h. A nitrocellulose ¢lter previously saturated in 10 mM IPTG (isopropyl L-Dthiogalactopyranoside) was placed onto the top agarose. The plate was incubated at 37³C for 3.5 h. The nitrocellulose ¢lter was removed, rinsed with a bu¡er containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween-20, blocked with 10% nonfat dry milk, and then incubated with 1:500 diluted rabbit anti-C. elegans microsome polyclonal antibody. Alkaline phosphatase-conjugated mouse anti-rabbit IgG (Sigma Chemical Co., St. Louis, MO) was diluted to 1:7000 for use as the secondary antibody. 5Bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (Bio-Rad Laboratories, Hercules, CA) was used for color development. Positive phage plaques were selected and rescreened at a low plaque density (100 plaques/90-mm plate). The puri¢ed plaques were picked with toothpicks and placed into tubes containing a PCR reaction mixture. PCR ampli¢cation of the insert cDNA was carried out without isolation of the phage DNA. The PCR protocol was the same as described previously [16] except that the annealing temperature was 58³C. PCR primers were designed from the Vgt11 vector upstream and downstream of the insert. The forward primer, Vgt11-A1, was 5P-GGATTGGTGGCGACGACT ; the reverse primer, Vgt11-A2, had the sequence 5P-GACATGGCCTGCCCGGTT. The ampli¢ed PCR products were puri¢ed from agarose gels by a gel extraction kit (Qiagen, Inc., Valencia, CA). Puri¢ed PCR
products were sequenced directly using primers Vgt11-A1 and Vgt11-A2. ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA) was used for sequencing, following the manufacturer's instructions. An automatic ABI Prism 310 sequencer (PE Applied Biosystems) was used for electrophoresis. For full insert sequence determination, the PCR products from this cDNA clone were subcloned into the pGEM-T Easy vector, following the manufacturer's instructions (Promega Co., Madison, WI). A QIAprep Spin Miniprep kit (Qiagen) was used for isolation of the plasmid. The isolated plasmid from the subclone was used for sequencing. After obtaining insert sequence information from both ends, new sequencing primers were designed from the sequences until all of the insert sequence had been sequenced from both strands. Oligonucleotide primers were purchased from Universal DNA, Inc. (Tigard, OR). After the highly conserved heme-binding region was found from the sequence, a heme probe for C. elegans was designed with the sequence 5P-TTCTCAAATGGTAACAGACAATGTATTGGT. The heme probe was labeled with a DIG oligo 3P-end labeling kit, following the manufacturer's instructions (Boehringer Mannheim Co., Indianapolis, IN) and used to rescreen the C. elegans cDNA library. A Plaque-lift hybridization procedure was used for the screening, following the DIG system user's guide for ¢lter hybridization. The DIG Easy Hyb from Boehringer was used as the hybridization solution, with prehybridizing for 2 h at 42³C and hybridizing overnight at 42³C. DNA sequence analysis, translation, and alignment with related genes and proteins were carried out using the computer programs Lasergene (DNASTAR, Inc., Madison, WI) and Align Plus (Scienti¢c Educational Software, State Line, PA). The Lasergene was used for the construction of the tree using the Clustal method. The GenBank program BLAST [17] was used to ¢nd similar sequences from GenBank. 2.3. Construction of a plasmid expressing His-tagged C. elegans P450 After the C. elegans P450 gene sequence was determined, the gene (without vector sequence) was ampli¢ed by PCR from the cDNA clone. Three primers were designed for the ampli¢cation of the C. elegans P450 gene. The forward primers, 450-F1-Bam and 450-F2-Bam, had the sequences cgggatccACAAATGGTTCAACTTGGAAA and cgggatccAATAATTTCATTTTTAAACGACTAAACGT, respectively. Capital letters in the sequence represent the starting sequence of the full C. elegans P450 gene or the 389-amino acid (aa) open reading frame (ORF) without the start codon, ATG. Lower-case letters in the sequence represent the site for BamHI digestion.
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The reverse primer, 450-R-Hind, had the sequence ccaagcttTTAATATCTTCTCTTGAATCGGATT. Capital letters in the sequence are complementary to the reverse sequence of the 3P end, including the stop codon of the C. elegans P450 gene. Lower-case letters represent the site for HindIII digestion. The PCR products were puri¢ed from agarose gels by a gel extraction kit (Qiagen) and digested with BamHI and HindIII. The PCR products were then ligated with BamHI- and HindIII-digested plasmid pQE30, a His-tagged expression vector for E. coli (Qiagen). E. coli strain SG13009 (Qiagen) was used as a host for the transformation and expression of the P450 gene. 2.4. SDS^PAGE analysis of the recombinant protein induced from the pQE30-CE-P450 clone Bacterial cells harboring the CE-P450 clone were cultured to OD600 = 0.7^0.9 in 100 ml LB containing ampicillin (100 Wg ml31 ) and kanamycin (25 Wg ml31 ). IPTG (1.5 mM) was added to induce expression of the recombinant protein and the culture was shaken continuously at 37³C for 5 h. After induction, the cells were harvested by centrifugation at 10 000Ug for 10 min at 4³C. The pellet was suspended in bu¡er B (8 M urea, 0.1 M Na-phosphate, 0.01 M Tris, pH 8.0) and freeze-thawed three times to break the cells. These were centrifuged again at 10 000Ug. Both the supernatant and the pellet, after treatment with SDS bu¡er, were analyzed by SDS^PAGE in a Mini-Protean II Dual Slab Cell (Bio-Rad). 2.5. Immunostaining of the recombinant P450 protein expressed from the pQE30-CE E. coli clone IPTG-induced cells from the pQE30-CE E. coli clone were lysed by sonication. The pellet was resuspended in SDS sample bu¡er, boiled for 5 min and centrifuged at 14 000Ug for 5 min. The supernatant was used for SDS^ PAGE and then transferred to a nitrocellulose membrane in a mini trans-blot electrophoretic transfer cell (Bio-Rad). The membrane was stained with 0.1% fast green (Bio-Rad) in 10% acetic acid and 50% methanol, then cut into strips to separate the sample lanes. Monoclonal antibody (mAb) against rat P450 1A1 (PM10, used for the identi¢cation of the polycyclic aromatic hydrocarbon induced P450 1A1), mAb against rat P450 3A1 (PM40, prepared against puri¢ed rat P450 3A1), and rat liver microsomes for positive control (AM10, prepared from male Fischer F344 rats and used as a control for P450 enzyme activity assays) were purchased from Oxford Biomedical Research, Inc. (Oxford, MI). A goat anti-rat CYP2E1 polyclonal antibody was purchased from Daichi Pure Chemical Co. (Tokyo). 2.6. Benzidine staining of hemoproteins Benzidine dihydrochloride was used to detect hemopro-
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teins in SDS gels following the method of Moore et al. [18]. 2.7. GenBank accession number The C. elegans P450 gene was deposited in the GenBank database with accession number AF249299. 3. Results and discussion We have shown earlier that the cytochrome P450 of C. elegans is involved in the metabolism and biotransformation of a number of xenobiotics [6^11]. In this paper, we present the cloning, sequencing, and expression of a cytochrome P450 gene of C. elegans. Initially, a cDNA library of C. elegans was constructed into the EcoRI site of the Vgt11 vector. The cDNA library was then screened with the help of polyclonal antibodies raised against C. elegans microsomes, where cytochrome P450 is localized [5]. Antibody screening yielded one positive clone. Nucleotide sequence analysis of the clone suggested the presence of an ORF encoding a 389-aa protein with MW 44 971.10. The amino acid homology search with the known GenBank database sequences found 506 matches, all belonging to the cytochrome P450 family of genes. All the cytochrome P450 genes contain a highly conserved heme-binding region, FxxGxxxCxG [19]. This conserved region is regarded as a ¢ngerprint for the identi¢cation of cytochrome P450 genes. Based upon this conserved region, Kullman and Matsumura [20] con¢rmed the presence of a cytochrome P450 gene in the fungus Phanerochaete chrysosporium. In order to demonstrate the relatedness of the 389-aa clone to cytochrome P450, an oligo probe corresponding to the heme-binding region was designed to rescreen the cDNA library of C. elegans. Two new clones were obtained. Sequence comparison of the two oligo-screened clones and a previous antibody-screened 389-aa clone showed that the three clones were closely related but had di¡erent length cDNA inserts. They were also from the same mRNA of the C. elegans P450 gene. The presence of heme-binding regions in the oligo- and antibodyscreened clones clearly indicated that the clones were closely related to cytochrome P450 (Fig. 1A). The truncated copies of the cytochrome P450 gene in some of the clones obtained from the cDNA library were probably due to incomplete reverse transcription from the mRNAs. The two oligo-screened clones contained the 389-aa coding sequence along with di¡erent length upstream sequences. One of the oligo-screened clones contained the same ORF for the 389 amino acids as the clone screened by the antibody, but had less of the 5P end sequence. The other oligo-screened clone contained a large ORF encoding a protein of 473 amino acids with MW 54 958.60 and
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Fig. 1. Comparison of the DNA sequences from the three clones (A), the deduced full C. elegans P450 sequence (B), and the clear picture of the DNA sequencing for the heme-binding region (C). The highly conserved heme-binding protein sequence, FxxGxxxCxG, is underlined in B. Clone 1, screened by antibody. Clones 2 and 3, screened by heme-oligo probe. Clone 3 contains the full C. elegans P450 gene. Dots indicate the same sequence as in the ¢rst line (clone 3). Dashes indicate that the sequence is lacking. The start locations for the 473 aa and 389 aa are indicated by underlines. The stop codon, TAA (underlined), is the same for the 473 and 389 aa. The 5P ends of the three DNA sequences are the adapter sequence used for cDNA library preparation.
represented the full-length cytochrome P450 gene of C. elegans (Fig. 1B,C). The full C. elegans P450 gene was successfully subcloned and overexpressed in a plasmid^E. coli system (pQE30). The overexpressed recombinant protein, upon analysis in SDS gels, migrated at 56 kDa (Fig. 2). Of the 56-kDa protein, 55 kDa was represented by the cloned gene and 1 kDa was represented by the 6UHis tag. Three methods were used to demonstrate that the recombinant protein in E. coli was a P450. The amino acid sequence was an identical to the DNA-deduced P450 sequence. Western blot analysis with three antibodies (CYP1A1, CYP2E1, and CYP3A1) showed that the recombinant protein in E. coli was homologous to all the mammalian P450s tested (Fig. 3B). Benzidine staining to detect hemoproteins in SDS gels gave positive results, indicating that the heme group was present (Fig. 3C). Since the recombinant protein was found in the pellet and not in the supernatant fraction, it showed low P450 activity when tested with cyclobenzaprine and phenanthrene (data not shown). Therefore, achievement of signi¢cant enzyme activity may require other components of the P450 monooxygenase that are essential to transfer electrons for catalytic reactions which are not optimally expressed using the E. coli system. A phylogenetic tree was constructed by comparison of
the C. elegans P450 with other fungal P450 protein sequences published in the GenBank. The C. elegans P450 clustered close to the cyp51 family, which includes Candida albicans, Ustilago maydis, Penicillium italicum, and Schizosaccharomyces pombe (Fig. 4), and was named cyp509A1 by the International Committee on the Nomen-
Fig. 2. SDS^polyacrylamide gel showing the overexpressed recombinant protein (56 kDa) from a subclone, pQE30-CE-P450 (473 aa) in E. coli. Lane M, molecular size marker. Lane 1, supernatant from 8 M urea bu¡er-treated cells. Lane 2, pellet from 8 M urea bu¡er-treated cells.
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Fig. 3. Immunostain and benzidine stain for P450. Lane M, molecular size marker. Lane 1, rat liver microsome preparation containing P450s at 50 kDa (positive control). Lane 2, E. coli proteins containing the overexpressed recombinant protein at 46 kDa from a subclone, pQE30-CE-389 aa, in E. coli. A: Coomassie blue-stained SDS gel for control to show the total proteins. B: Three P450 antibody-stained membranes with proteins transferred from the SDS gel. The three antibodies are CYP2E1, CYP1A1, and CYP3A1, which were described in Section 2. C: Benzidine-stained SDS gel.
clature for Cytochrome P450 Enzymes. Nelson [21] has suggested that a common ancestor to fungi and animals had only the cyp51 P450 gene, which encodes sterol 14Kdemethylase. He also indicated that this enzyme may have been the ¢rst eukaryotic P450 [22]. Here we show for the ¢rst time that the zygomycete C. elegans P450 clusters with fungi in that cyp51 family and has immunological
homology to some mammalian cytochrome P450s [23]. Our previous observations showed that the phase I reactions are catalyzed by cytochrome P450 in C. elegans [5,11]. The successful cloning and expression of the P450 gene described in this paper would further assist in understanding the mechanism of biotransformation and detoxi¢cation reactions in C. elegans.
Fig. 4. Phylogenetic tree constructed by comparison of the C. elegans P450 (473 aa) with other fungal P450 protein sequences published in the GenBank. The scale indicates the amino acid di¡erence.
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In conclusion, a full-length cDNA clone of C. elegans cytochrome P450 was cloned and characterized. It was subcloned and overexpressed in a plasmid^E. coli system (pQE30). The C. elegans cytochrome P450 gene clustered close to cyp51 in a phylogenetic tree and was named cyp509A1. The results provide more evidence of the feasibility of using C. elegans for modeling the mammalian metabolism of pharmaceutically important compounds. Acknowledgements We appreciate Dr. David R. Nelson of the International Committee on the Nomenclature for Cytochrome P450 Enzymes for stimulating discussion and classifying the C. elegans P450 as cyp509A1. We also thank Drs. Saeed A. Khan and John B. Sutherland for critically reading the manuscript and Ms. Pat Fleischer for clerical assistance. A part of this work was supported by US Environmental Protection Agency Cooperative Agreement CR 820773. References [1] Azerad, R. (1999) Microbial models for drug metabolism. In: Advances in Biochemical Engineering/Biotechnology (Scheper, T., Ed.), Vol. 63, pp. 169^218. Springer-Verlag, Berlin. [2] Cerniglia, C.E. (1997) Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation. J. Ind. Microbiol. Biotechnol. 19, 324^333. [3] Davis, P.J. (1988) Microbial models of mammalian drug metabolism. Dev. Ind. Microbiol. 29, 197^219. [4] Rao, G.P. and Davis, P.J. (1997) Microbial models of mammalian metabolism: Biotransformations of HP 749 (Besipiridine) using Cunninghamella elegans. Drug Metab. Dispos. 25, 709^715. [5] Zhang, D.L., Yang, Y., Leakey, J.E.A. and Cerniglia, C.E. (1996) Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics. FEMS Microbiol. Lett. 138, 221^ 226. [6] Hansen Jr., E.B., Cho, B.P., Korfmacher, W.A. and Cerniglia, C.E. (1995) Fungal transformations of antihistamines : metabolism of brompheniramine, chlorpheniramine, and pheniramine to N-oxide and N-demethylated metabolites by the fungus Cunninghamella elegans. Xenobiotica 25, 1081^1092. [7] Moody, J.D., Freeman, J.P. and Cerniglia, C.E. (1999) Biotransformation of doxepin by Cunninghamella elegans. Drug Metab. Dispos. 27, 1157^1164.
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[8] Zhang, D.L., Evans, F.E., Freeman, J.P., Duhart Jr., B.T. and Cerniglia, C.E. (1995) Biotransformation of amitriptyline by Cunninghamella elegans. Drug Metab. Dispos. 23, 1417^1425. [9] Zhang, D.L., Evans, F.E., Freeman, J.P., Yang, Y., Deck, J. and Cerniglia, C.E. (1996) Formation of mammalian metabolites of cyclobenzaprine by the fungus, Cunninghamella elegans. Chem.-Biol. Interact. 102, 79^92. [10] Zhang, D.L., Freeman, J.P., Sutherland, J.B., Walker, A.E., Yang, Y. and Cerniglia, C.E. (1996) Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans. Appl. Environ. Microbiol. 62, 798^803. [11] Zhang, D.L., Hansen, E.B., Deck, J., Heinze, T.M., Sutherland, J.B. and Cerniglia, C.E. (1996) Fungal biotransformation of the antihistamine azatadine by Cunninghamella elegans. Appl. Environ. Microbiol. 62, 3477^3479. [12] Wang, R.-F., Khan, A.A., Cao, W.-W. and Cerniglia, C.E. (2000) Cloning, sequencing, and expression of the gene encoding enolase from a fungus, Cunninghamella elegans. Mycol. Res. 104, 175^179. [13] Wang, R.-F., Khan, A.A., Cao, W.-W. and Cerniglia, C.E. (1998) Identi¢cation and sequencing of the gene encoding 6-phosphogluconate dehydrogenase from a fungus, Cunninghamella elegans and expression of the gene in E. coli. FEMS Microbiol. Lett. 169, 397^ 402. [14] van den Brink, H.J.M., van Gorcom, R.F.M., van den Hondel, C.A.M.J.J. and Punt, P.J. (1998) Review ^ Cytochrome P450 enzyme systems in fungi. Fungal Genet. Biol. 23, 1^17. [15] Maniatis, T., Fritsch, E. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [16] Wang, R.-F., Cao, W.-W. and Cerniglia, C.E. (1996) PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl. Environ. Microbiol. 62, 1242^1247. [17] Altschul, S.F., Madden, T.L., Scha«¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402. [18] Moore, R.W., Welton, A.F. and Aust, S.D. (1978) Detection of hemoproteins in SDS-polyacrylamide gels. Methods Enzymol. 52, 324^ 331. [19] Gonzalez, F.J. (1989) The molecular biology of cytochrome P450s. Pharmacol. Res. 40, 243^288. [20] Kullman, S.W. and Matsumura, F. (1997) Identi¢cation of a novel cytochrome P450 gene from the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 63, 2741^2746. [21] Nelson, D.R. (1998) Metazoan cytochrome P450 evolution. Comp. Biochem. Physiol. C 121, 15^22. [22] Nelson, D.R. (1999) Cytochrome P450 and the individuality of species. Arch. Biochem. Biophys. 369, 1^10. [23] Parkinson, A. (1996) An overview of current cytochrome P450 technology for assessing the safety and e¤cacy of new materials. Toxicol. Pathol. 24, 45^57.
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Cloning, sequencing, and expression in Escherichia coli of a cytochrome P450 gene from Cunninghamella elegans Rong-Fu Wang, Wei-Wen Cao, Ashraf A. Khan, Carl E. Cerniglia * Division of Microbiology, National Center for Toxicological Research, FDA, Je¡erson, AR 72079-9502, USA Received 22 November 1999; received in revised form 8 May 2000; accepted 8 May 2000
Abstract A polyclonal antibody against microsomes of a fungus, Cunninghamella elegans, was used to screen a C. elegans cDNA library. A cDNA clone, containing an open reading frame (ORF) encoding a protein of 389 amino acids (aa), was obtained. GenBank comparison (BLAST) showed that the protein was closely related to P450 because a heme-binding region, which is highly conserved in all P450 sequences, was found in the ORF protein. Using an oligo probe designed from this C. elegans heme-binding region to rescreen the cDNA library, we obtained three new clones. Sequence comparison showed that the three clones, with different length cDNA inserts, were from the same mRNA of the C. elegans P450 gene. One clone had the full C. elegans P450 gene, encoding 473 aa with a molecular mass of 54 958.60, whereas the 389 was a part of the 473 aa without the N-terminal. The entire C. elegans P450 gene was successfully subcloned and overexpressed in a plasmid^Escherichia coli system (pQE30). Immunostaining with three antibodies (CYP1A1, CYP2E1, and CYP3A1) against mammalian P450 enzymes and benzidine staining for hemoproteins showed positive results for the recombinant protein expressed in E. coli. A phylogenetic tree was constructed by comparison of other fungal P450s to the C. elegans sequence. The C. elegans P450 clustered close to the cyp51 family and was named cyp509A1 by the International Committee on the Nomenclature for Cytochrome P450 Enzymes. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : P450; Cloning; Expression ; Cunninghamella elegans
1. Introduction The ¢lamentous fungus, Cunninghamella elegans, is a zygomycete, which has been used as a microbial model of mammalian metabolism and for the biodegradation of priority pollutants [1^4]. C. elegans ATCC 36112 metabolizes or biotransforms numerous structurally diverse compounds, such as polycyclic aromatic hydrocarbons (PAHs), crude oil components, drugs, and N-,S-,O-heterocyclic aromatic compounds, by phase I and phase II enzymes [5]. The phase I reactions catalyzed by cytochrome P450 in C. elegans include aliphatic and aromatic hydroxylation, N- and O-dealkylation and N- and S-oxidation [5^ 11]. The involvement of cytochrome P450 enzymes in the metabolism of PAHs and pharmaceutical compounds by C. elegans is based on carbon monoxide di¡erence spectra (absorption at 450 nm), enzyme activities, cytochrome P450 inhibitors, 18 O2 incorporation experiments,
and spectra of metabolites that are similar to those formed by mammalian cytochrome P450 enzyme systems [2,8,9]. Little is known about the molecular genetics of C. elegans, except for studies of the enolase and the 6-phosphogluconate dehydrogenase conducted in this laboratory [12,13]. The use of molecular biology techniques to characterize fungal cytochrome P450 enzyme systems has greatly enhanced our understanding of fungal biotransformation of xenobiotics, especially since it is di¤cult to purify fungal microsomal P450 proteins using conventional biochemical techniques [14]. Since many of the phase I and phase II enzymes are localized in the microsomal fraction of the cell [5], a polyclonal antibody against C. elegans microsomes was used to screen a C. elegans cDNA library in this study. The isolation and characterization of three cDNA clones encoding a C. elegans cytochrome P450 is reported in this article.
* Corresponding author. Tel. : +1 (870) 543-7341; Fax: +1 (870) 543-7307; E-mail: [email protected] 0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 2 1 0 - X
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2. Materials and methods 2.1. Mycelia, microsomes, cDNA library and rabbit antiserum preparations C. elegans ATCC 36112 was grown in Sabouraud dextrose broth (Difco Laboratories, Detroit, MI) at room temperature without shaking. After 5 days of incubation, the mycelium was harvested by ¢ltration and washed with deionized water. Freshly prepared C. elegans mycelium was frozen in liquid nitrogen and sent on dry ice to Clontech Laboratories, Inc. (Palo Alto, CA) for custom mRNA preparation and custom cDNA library construction. Microsomes isolated from C. elegans mycelia [5] were sent to Alpha Diagnostic (San Antonio, TX) for custom antiserum preparation from rabbit (the antibody titer was 104 ). 2.2. Screening the cDNA library, ampli¢cation and DNA sequence analysis Rabbit antiserum raised to C. elegans microsomes was used to screen the unampli¢ed C. elegans cDNA library. The cDNA library (5U103 /90-mm plate) was mixed with Escherichia coli Y1090, which had been cultured in LB broth [15]^0.2% maltose^10 mM MgSO4 . After 20 min adsorption, 3 ml LB-soft top agarose (0.6%) at 55³C was added and immediately poured onto an LB agar plate. The plate was incubated at 42³C for 3.5 h. A nitrocellulose ¢lter previously saturated in 10 mM IPTG (isopropyl L-Dthiogalactopyranoside) was placed onto the top agarose. The plate was incubated at 37³C for 3.5 h. The nitrocellulose ¢lter was removed, rinsed with a bu¡er containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween-20, blocked with 10% nonfat dry milk, and then incubated with 1:500 diluted rabbit anti-C. elegans microsome polyclonal antibody. Alkaline phosphatase-conjugated mouse anti-rabbit IgG (Sigma Chemical Co., St. Louis, MO) was diluted to 1:7000 for use as the secondary antibody. 5Bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (Bio-Rad Laboratories, Hercules, CA) was used for color development. Positive phage plaques were selected and rescreened at a low plaque density (100 plaques/90-mm plate). The puri¢ed plaques were picked with toothpicks and placed into tubes containing a PCR reaction mixture. PCR ampli¢cation of the insert cDNA was carried out without isolation of the phage DNA. The PCR protocol was the same as described previously [16] except that the annealing temperature was 58³C. PCR primers were designed from the Vgt11 vector upstream and downstream of the insert. The forward primer, Vgt11-A1, was 5P-GGATTGGTGGCGACGACT ; the reverse primer, Vgt11-A2, had the sequence 5P-GACATGGCCTGCCCGGTT. The ampli¢ed PCR products were puri¢ed from agarose gels by a gel extraction kit (Qiagen, Inc., Valencia, CA). Puri¢ed PCR
products were sequenced directly using primers Vgt11-A1 and Vgt11-A2. ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA) was used for sequencing, following the manufacturer's instructions. An automatic ABI Prism 310 sequencer (PE Applied Biosystems) was used for electrophoresis. For full insert sequence determination, the PCR products from this cDNA clone were subcloned into the pGEM-T Easy vector, following the manufacturer's instructions (Promega Co., Madison, WI). A QIAprep Spin Miniprep kit (Qiagen) was used for isolation of the plasmid. The isolated plasmid from the subclone was used for sequencing. After obtaining insert sequence information from both ends, new sequencing primers were designed from the sequences until all of the insert sequence had been sequenced from both strands. Oligonucleotide primers were purchased from Universal DNA, Inc. (Tigard, OR). After the highly conserved heme-binding region was found from the sequence, a heme probe for C. elegans was designed with the sequence 5P-TTCTCAAATGGTAACAGACAATGTATTGGT. The heme probe was labeled with a DIG oligo 3P-end labeling kit, following the manufacturer's instructions (Boehringer Mannheim Co., Indianapolis, IN) and used to rescreen the C. elegans cDNA library. A Plaque-lift hybridization procedure was used for the screening, following the DIG system user's guide for ¢lter hybridization. The DIG Easy Hyb from Boehringer was used as the hybridization solution, with prehybridizing for 2 h at 42³C and hybridizing overnight at 42³C. DNA sequence analysis, translation, and alignment with related genes and proteins were carried out using the computer programs Lasergene (DNASTAR, Inc., Madison, WI) and Align Plus (Scienti¢c Educational Software, State Line, PA). The Lasergene was used for the construction of the tree using the Clustal method. The GenBank program BLAST [17] was used to ¢nd similar sequences from GenBank. 2.3. Construction of a plasmid expressing His-tagged C. elegans P450 After the C. elegans P450 gene sequence was determined, the gene (without vector sequence) was ampli¢ed by PCR from the cDNA clone. Three primers were designed for the ampli¢cation of the C. elegans P450 gene. The forward primers, 450-F1-Bam and 450-F2-Bam, had the sequences cgggatccACAAATGGTTCAACTTGGAAA and cgggatccAATAATTTCATTTTTAAACGACTAAACGT, respectively. Capital letters in the sequence represent the starting sequence of the full C. elegans P450 gene or the 389-amino acid (aa) open reading frame (ORF) without the start codon, ATG. Lower-case letters in the sequence represent the site for BamHI digestion.
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The reverse primer, 450-R-Hind, had the sequence ccaagcttTTAATATCTTCTCTTGAATCGGATT. Capital letters in the sequence are complementary to the reverse sequence of the 3P end, including the stop codon of the C. elegans P450 gene. Lower-case letters represent the site for HindIII digestion. The PCR products were puri¢ed from agarose gels by a gel extraction kit (Qiagen) and digested with BamHI and HindIII. The PCR products were then ligated with BamHI- and HindIII-digested plasmid pQE30, a His-tagged expression vector for E. coli (Qiagen). E. coli strain SG13009 (Qiagen) was used as a host for the transformation and expression of the P450 gene. 2.4. SDS^PAGE analysis of the recombinant protein induced from the pQE30-CE-P450 clone Bacterial cells harboring the CE-P450 clone were cultured to OD600 = 0.7^0.9 in 100 ml LB containing ampicillin (100 Wg ml31 ) and kanamycin (25 Wg ml31 ). IPTG (1.5 mM) was added to induce expression of the recombinant protein and the culture was shaken continuously at 37³C for 5 h. After induction, the cells were harvested by centrifugation at 10 000Ug for 10 min at 4³C. The pellet was suspended in bu¡er B (8 M urea, 0.1 M Na-phosphate, 0.01 M Tris, pH 8.0) and freeze-thawed three times to break the cells. These were centrifuged again at 10 000Ug. Both the supernatant and the pellet, after treatment with SDS bu¡er, were analyzed by SDS^PAGE in a Mini-Protean II Dual Slab Cell (Bio-Rad). 2.5. Immunostaining of the recombinant P450 protein expressed from the pQE30-CE E. coli clone IPTG-induced cells from the pQE30-CE E. coli clone were lysed by sonication. The pellet was resuspended in SDS sample bu¡er, boiled for 5 min and centrifuged at 14 000Ug for 5 min. The supernatant was used for SDS^ PAGE and then transferred to a nitrocellulose membrane in a mini trans-blot electrophoretic transfer cell (Bio-Rad). The membrane was stained with 0.1% fast green (Bio-Rad) in 10% acetic acid and 50% methanol, then cut into strips to separate the sample lanes. Monoclonal antibody (mAb) against rat P450 1A1 (PM10, used for the identi¢cation of the polycyclic aromatic hydrocarbon induced P450 1A1), mAb against rat P450 3A1 (PM40, prepared against puri¢ed rat P450 3A1), and rat liver microsomes for positive control (AM10, prepared from male Fischer F344 rats and used as a control for P450 enzyme activity assays) were purchased from Oxford Biomedical Research, Inc. (Oxford, MI). A goat anti-rat CYP2E1 polyclonal antibody was purchased from Daichi Pure Chemical Co. (Tokyo). 2.6. Benzidine staining of hemoproteins Benzidine dihydrochloride was used to detect hemopro-
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teins in SDS gels following the method of Moore et al. [18]. 2.7. GenBank accession number The C. elegans P450 gene was deposited in the GenBank database with accession number AF249299. 3. Results and discussion We have shown earlier that the cytochrome P450 of C. elegans is involved in the metabolism and biotransformation of a number of xenobiotics [6^11]. In this paper, we present the cloning, sequencing, and expression of a cytochrome P450 gene of C. elegans. Initially, a cDNA library of C. elegans was constructed into the EcoRI site of the Vgt11 vector. The cDNA library was then screened with the help of polyclonal antibodies raised against C. elegans microsomes, where cytochrome P450 is localized [5]. Antibody screening yielded one positive clone. Nucleotide sequence analysis of the clone suggested the presence of an ORF encoding a 389-aa protein with MW 44 971.10. The amino acid homology search with the known GenBank database sequences found 506 matches, all belonging to the cytochrome P450 family of genes. All the cytochrome P450 genes contain a highly conserved heme-binding region, FxxGxxxCxG [19]. This conserved region is regarded as a ¢ngerprint for the identi¢cation of cytochrome P450 genes. Based upon this conserved region, Kullman and Matsumura [20] con¢rmed the presence of a cytochrome P450 gene in the fungus Phanerochaete chrysosporium. In order to demonstrate the relatedness of the 389-aa clone to cytochrome P450, an oligo probe corresponding to the heme-binding region was designed to rescreen the cDNA library of C. elegans. Two new clones were obtained. Sequence comparison of the two oligo-screened clones and a previous antibody-screened 389-aa clone showed that the three clones were closely related but had di¡erent length cDNA inserts. They were also from the same mRNA of the C. elegans P450 gene. The presence of heme-binding regions in the oligo- and antibodyscreened clones clearly indicated that the clones were closely related to cytochrome P450 (Fig. 1A). The truncated copies of the cytochrome P450 gene in some of the clones obtained from the cDNA library were probably due to incomplete reverse transcription from the mRNAs. The two oligo-screened clones contained the 389-aa coding sequence along with di¡erent length upstream sequences. One of the oligo-screened clones contained the same ORF for the 389 amino acids as the clone screened by the antibody, but had less of the 5P end sequence. The other oligo-screened clone contained a large ORF encoding a protein of 473 amino acids with MW 54 958.60 and
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Fig. 1. Comparison of the DNA sequences from the three clones (A), the deduced full C. elegans P450 sequence (B), and the clear picture of the DNA sequencing for the heme-binding region (C). The highly conserved heme-binding protein sequence, FxxGxxxCxG, is underlined in B. Clone 1, screened by antibody. Clones 2 and 3, screened by heme-oligo probe. Clone 3 contains the full C. elegans P450 gene. Dots indicate the same sequence as in the ¢rst line (clone 3). Dashes indicate that the sequence is lacking. The start locations for the 473 aa and 389 aa are indicated by underlines. The stop codon, TAA (underlined), is the same for the 473 and 389 aa. The 5P ends of the three DNA sequences are the adapter sequence used for cDNA library preparation.
represented the full-length cytochrome P450 gene of C. elegans (Fig. 1B,C). The full C. elegans P450 gene was successfully subcloned and overexpressed in a plasmid^E. coli system (pQE30). The overexpressed recombinant protein, upon analysis in SDS gels, migrated at 56 kDa (Fig. 2). Of the 56-kDa protein, 55 kDa was represented by the cloned gene and 1 kDa was represented by the 6UHis tag. Three methods were used to demonstrate that the recombinant protein in E. coli was a P450. The amino acid sequence was an identical to the DNA-deduced P450 sequence. Western blot analysis with three antibodies (CYP1A1, CYP2E1, and CYP3A1) showed that the recombinant protein in E. coli was homologous to all the mammalian P450s tested (Fig. 3B). Benzidine staining to detect hemoproteins in SDS gels gave positive results, indicating that the heme group was present (Fig. 3C). Since the recombinant protein was found in the pellet and not in the supernatant fraction, it showed low P450 activity when tested with cyclobenzaprine and phenanthrene (data not shown). Therefore, achievement of signi¢cant enzyme activity may require other components of the P450 monooxygenase that are essential to transfer electrons for catalytic reactions which are not optimally expressed using the E. coli system. A phylogenetic tree was constructed by comparison of
the C. elegans P450 with other fungal P450 protein sequences published in the GenBank. The C. elegans P450 clustered close to the cyp51 family, which includes Candida albicans, Ustilago maydis, Penicillium italicum, and Schizosaccharomyces pombe (Fig. 4), and was named cyp509A1 by the International Committee on the Nomen-
Fig. 2. SDS^polyacrylamide gel showing the overexpressed recombinant protein (56 kDa) from a subclone, pQE30-CE-P450 (473 aa) in E. coli. Lane M, molecular size marker. Lane 1, supernatant from 8 M urea bu¡er-treated cells. Lane 2, pellet from 8 M urea bu¡er-treated cells.
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Fig. 3. Immunostain and benzidine stain for P450. Lane M, molecular size marker. Lane 1, rat liver microsome preparation containing P450s at 50 kDa (positive control). Lane 2, E. coli proteins containing the overexpressed recombinant protein at 46 kDa from a subclone, pQE30-CE-389 aa, in E. coli. A: Coomassie blue-stained SDS gel for control to show the total proteins. B: Three P450 antibody-stained membranes with proteins transferred from the SDS gel. The three antibodies are CYP2E1, CYP1A1, and CYP3A1, which were described in Section 2. C: Benzidine-stained SDS gel.
clature for Cytochrome P450 Enzymes. Nelson [21] has suggested that a common ancestor to fungi and animals had only the cyp51 P450 gene, which encodes sterol 14Kdemethylase. He also indicated that this enzyme may have been the ¢rst eukaryotic P450 [22]. Here we show for the ¢rst time that the zygomycete C. elegans P450 clusters with fungi in that cyp51 family and has immunological
homology to some mammalian cytochrome P450s [23]. Our previous observations showed that the phase I reactions are catalyzed by cytochrome P450 in C. elegans [5,11]. The successful cloning and expression of the P450 gene described in this paper would further assist in understanding the mechanism of biotransformation and detoxi¢cation reactions in C. elegans.
Fig. 4. Phylogenetic tree constructed by comparison of the C. elegans P450 (473 aa) with other fungal P450 protein sequences published in the GenBank. The scale indicates the amino acid di¡erence.
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In conclusion, a full-length cDNA clone of C. elegans cytochrome P450 was cloned and characterized. It was subcloned and overexpressed in a plasmid^E. coli system (pQE30). The C. elegans cytochrome P450 gene clustered close to cyp51 in a phylogenetic tree and was named cyp509A1. The results provide more evidence of the feasibility of using C. elegans for modeling the mammalian metabolism of pharmaceutically important compounds. Acknowledgements We appreciate Dr. David R. Nelson of the International Committee on the Nomenclature for Cytochrome P450 Enzymes for stimulating discussion and classifying the C. elegans P450 as cyp509A1. We also thank Drs. Saeed A. Khan and John B. Sutherland for critically reading the manuscript and Ms. Pat Fleischer for clerical assistance. A part of this work was supported by US Environmental Protection Agency Cooperative Agreement CR 820773. References [1] Azerad, R. (1999) Microbial models for drug metabolism. In: Advances in Biochemical Engineering/Biotechnology (Scheper, T., Ed.), Vol. 63, pp. 169^218. Springer-Verlag, Berlin. [2] Cerniglia, C.E. (1997) Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation. J. Ind. Microbiol. Biotechnol. 19, 324^333. [3] Davis, P.J. (1988) Microbial models of mammalian drug metabolism. Dev. Ind. Microbiol. 29, 197^219. [4] Rao, G.P. and Davis, P.J. (1997) Microbial models of mammalian metabolism: Biotransformations of HP 749 (Besipiridine) using Cunninghamella elegans. Drug Metab. Dispos. 25, 709^715. [5] Zhang, D.L., Yang, Y., Leakey, J.E.A. and Cerniglia, C.E. (1996) Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics. FEMS Microbiol. Lett. 138, 221^ 226. [6] Hansen Jr., E.B., Cho, B.P., Korfmacher, W.A. and Cerniglia, C.E. (1995) Fungal transformations of antihistamines : metabolism of brompheniramine, chlorpheniramine, and pheniramine to N-oxide and N-demethylated metabolites by the fungus Cunninghamella elegans. Xenobiotica 25, 1081^1092. [7] Moody, J.D., Freeman, J.P. and Cerniglia, C.E. (1999) Biotransformation of doxepin by Cunninghamella elegans. Drug Metab. Dispos. 27, 1157^1164.
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[8] Zhang, D.L., Evans, F.E., Freeman, J.P., Duhart Jr., B.T. and Cerniglia, C.E. (1995) Biotransformation of amitriptyline by Cunninghamella elegans. Drug Metab. Dispos. 23, 1417^1425. [9] Zhang, D.L., Evans, F.E., Freeman, J.P., Yang, Y., Deck, J. and Cerniglia, C.E. (1996) Formation of mammalian metabolites of cyclobenzaprine by the fungus, Cunninghamella elegans. Chem.-Biol. Interact. 102, 79^92. [10] Zhang, D.L., Freeman, J.P., Sutherland, J.B., Walker, A.E., Yang, Y. and Cerniglia, C.E. (1996) Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans. Appl. Environ. Microbiol. 62, 798^803. [11] Zhang, D.L., Hansen, E.B., Deck, J., Heinze, T.M., Sutherland, J.B. and Cerniglia, C.E. (1996) Fungal biotransformation of the antihistamine azatadine by Cunninghamella elegans. Appl. Environ. Microbiol. 62, 3477^3479. [12] Wang, R.-F., Khan, A.A., Cao, W.-W. and Cerniglia, C.E. (2000) Cloning, sequencing, and expression of the gene encoding enolase from a fungus, Cunninghamella elegans. Mycol. Res. 104, 175^179. [13] Wang, R.-F., Khan, A.A., Cao, W.-W. and Cerniglia, C.E. (1998) Identi¢cation and sequencing of the gene encoding 6-phosphogluconate dehydrogenase from a fungus, Cunninghamella elegans and expression of the gene in E. coli. FEMS Microbiol. Lett. 169, 397^ 402. [14] van den Brink, H.J.M., van Gorcom, R.F.M., van den Hondel, C.A.M.J.J. and Punt, P.J. (1998) Review ^ Cytochrome P450 enzyme systems in fungi. Fungal Genet. Biol. 23, 1^17. [15] Maniatis, T., Fritsch, E. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [16] Wang, R.-F., Cao, W.-W. and Cerniglia, C.E. (1996) PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl. Environ. Microbiol. 62, 1242^1247. [17] Altschul, S.F., Madden, T.L., Scha«¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402. [18] Moore, R.W., Welton, A.F. and Aust, S.D. (1978) Detection of hemoproteins in SDS-polyacrylamide gels. Methods Enzymol. 52, 324^ 331. [19] Gonzalez, F.J. (1989) The molecular biology of cytochrome P450s. Pharmacol. Res. 40, 243^288. [20] Kullman, S.W. and Matsumura, F. (1997) Identi¢cation of a novel cytochrome P450 gene from the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 63, 2741^2746. [21] Nelson, D.R. (1998) Metazoan cytochrome P450 evolution. Comp. Biochem. Physiol. C 121, 15^22. [22] Nelson, D.R. (1999) Cytochrome P450 and the individuality of species. Arch. Biochem. Biophys. 369, 1^10. [23] Parkinson, A. (1996) An overview of current cytochrome P450 technology for assessing the safety and e¤cacy of new materials. Toxicol. Pathol. 24, 45^57.
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