Cloning, Expression and Functional Characterization of Cytochrome

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Cloning, Expression and Functional Characterization of Cytochrome P450 3A37 from Turkey Liver with High Aflatoxin B1 Epoxidation Activity† Sumit Rawal, Shirley S. M. Yip,‡ and Roger A. Coulombe, Jr.* Department of Veterinary Sciences, Graduate Program in Toxicology, Utah State UniVersity, Logan, Utah 84322-4620 ReceiVed January 26, 2010

Cytochrome P450s (P450) play an important role in the formation of carcinogenic and mutagenic electrophilic intermediates from a wide range of xenobiotics, including naturally occurring dietary compounds. The pathogenesis of hepatotoxic and hepatocarcinogenic action of the mycotoxin aflatoxin B1 (AFB1) involves initial bioactivation by P450s to a reactive and electrophilic intermediate exo-aflatoxin B1-8,9-epoxide (exo-AFBO). Poultry, especially turkeys are extremely sensitive to AFB1, a condition due, in part, to efficient epoxidation by P450 1A and 3A enzymes. We previously reported the discovery of P450 1A5 from turkey liver, which like its human homologue, 1A2, bioactivated AFB1 to exo-AFBO and aflatoxin M1 (AFM1). Here, we describe P450 3A37, the 3A4 homologue from turkey liver. This gene has an open reading frame (ORF) of 1512 bp, and the protein is predicted to be 504 amino acids with 97% identity to chicken P450 3A37. A truncated construct of the turkey P450 3A37 gene with 11 amino acids deleted from the hydrophobic N-terminal region was heterologously expressed in Escherichia coli, the protein from which exhibited a CO difference spectrum typical of P450s. Like human P450 3A4, 3A37 biotransformed AFB1 to exo-AFBO and aflatoxin Q1 (AFQ1) and possessed nifedipine oxidation activity, both of which were inhibited by the P450 3A4 inhibitor 17R-ethynylestradiol. Oxidation of AFB1 to exo-AFBO and AFQ1 by P450 3A37 followed sigmoidal Hill kinetics, suggestive of an allosteric interaction between the enzyme and AFB1. The Hill coefficient (n) value was 1.9 for exo-AFBO and 1.6 for AFQ1, indicative of positive cooperativity. The calculated Km and Vmax values for the formation of exo-AFBO were 287 ( 21 µM and 1.45 ( 0.07 nmol/min/nmol P450, respectively, whereas those of AFQ1 formation were 302 ( 51 µM and 7.86 ( 0.75 nmol/min/nmol P450, respectively. These data strongly suggest that P450 3A37, along with P450 1A5, plays an important role in AFB1 epoxidation in turkey liver. Introduction Cytochrome P450s (P4501) are the major drug metabolism enzymes involved in the metabolism of variety of endogenous and exogenous substrates (1). While converting most of the compounds into the readily excretable detoxified hydrophilic form, P450s also contribute to the toxicity of some xenobiotics through bioactivation, resulting in the formation of reactive intermediates which causes toxicity (2). The carcinogenic † GenBank accession numbers for the CYP3 A enzymes cited in this article are as follows: human CYP3A4, BC069418; Bos taurus (cattle), NM_174531; Cercopithecus aethiops (African green monkey), DQ022198; Mus musculus (house mouse), AB039380; Macaca mulatta (rhesus monkey), AY635466; Oncorhynchus mykiss (rainbow trout), AF267126. The nucleotide sequence for the turkey CYP3A37 gene has been deposited in the GenBank database under GenBank accession number DQ450083. The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number ABE28024. * To whom correspondence should be addressed. Tel: 435-797-1598. Fax: 435-797-1601. E-mail: [email protected]. ‡ Present address: Early Stage Cell Culture, Genentech, Inc., South San Francisco, CA 94080. 1 Abbreviations: AFB1, aflatoxin B1; AFBO, AFB1-8,9 epoxide; AFBOGSH, AFB1-8,9-epoxide glutathione conjugate; AFG1, aflatoxin G1; AFM1, aflatoxin M1; AFQ1, aflatoxin Q1; BHA, butylated hydroxyanisole; DMSO, dimethyl sulfoxide; endo-AFBO, endo-AFB1-8,9-epoxide; exo-AFBO, exoAFB1-8,9-epoxide; GST, glutathione S-transferase; hNPR, human NADPHP450 reductase; IPTG, isopropyl-β-D-thiogalactopyranoside; NCBI, National Center for Biotechnology Information; ORF, open reading frame; RACE, rapid amplification of cDNA ends; UTR, untranslated region.

mycotoxin aflatoxin B1 (AFB1) is bioactivated by P450s to the electrophilic and highly reactive epoxide intermediate exo-AFB18,9-epoxide (exo-AFBO) (2-5), which binds to DNA, RNA, and other cellular proteins and is responsible for mediating the toxic and carcinogenic effects of AFB1 in humans as well as most animal species. In many species, glutathione-S-transferases (GSTs) detoxify the exo-AFBO so produced by conjugating it with endogenous glutathione. Poultry, specifically turkeys, are extremely sensitive to aflatoxicosis (6-11). The susceptibility of turkeys to AFB1 was first demonstrated in the early 1960s as the etiological agent of Turkey X disease, which caused widespread deaths of turkeys and other poultry in Europe (12). Aflatoxins are ubiquitous in corn-based animal feeds, their presence in feed cause significant economic losses to the poultry industry every year, and contamination is practically unavoidable (13, 14). We have previously shown that this extreme sensitivity of turkeys to AFB1 is associated with efficient oxidation mediated by homologues to human P450 1A2 and 3A4, coupled with deficient detoxification by GSTs (10, 15, 16). Turkey liver possesses P450 1A5 (15), which, like its human counterpart, oxidized AFB1 to exo-AFBO and aflatoxin M1 (AFM1). Although both P450 1A and 3A enzymes oxidize AFB1, there are conflicting reports on their relative importance (5, 17-19). In human liver, P450 3A4, which appears to account for the

10.1021/tx1000267  2010 American Chemical Society Published on Web 07/12/2010

Turkey Hepatic P450 3A37

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Figure 1. Expression constructs of P450 3A37 showing nucleic acid and amino acid sequences at the N- and C-terminals. The wild type (wtP450 1A5) has 1512 bp encoding 504 amino acids; the truncated construct (tP450 1A5) has 1479 bp encoding 493 amino acids. Six histidine residues were added at the C-terminals to facilitate detection and purification. Nucleotides and amino acids different from the wild type sequence are bold faced.

majority of the formation of exo-AFBO and AFQ1, was an important determinant of AFB1 disposition (19, 20). We recently elucidated the genetic structure of turkey P450 3A37, which like human P450 3A4, is organized into 13 exons and 12 introns (21). The exons of the turkey gene were 124, 94, 53, 100, 114, 89, 149, 128, 73, 161, 227, 163, and 475 bases in length. Exons 1 and 13 contained 71 and 93 coding nucleotides, respectively. Because of its presumed role in AFB1 bioactivation, we describe here the heterologous expression and functional characterization of this P450 3A4 homologue, 3A37.

Experimental Procedures Chemicals and Reagents. QIAprep spin miniprep kit, QIAquick PCR purification kit, and gel extraction kit were obtained from Qiagen (Valencia, CA). TOPO TA cloning kit and maximum efficiency DH5RF′IQ Escherichia coli cells were from Invitrogen (Carlsbad, CA). Restriction enzymes and T4 DNA ligase were from Fermentas (Hanover, MD). Exo- and endo-AFB1-8,9-epoxide glutathione conjugate (AFBO-GSH) standard were a generous gift from Dr. F. Peter Guengerich, Vanderbilt University School of Medicine (Nashville, TN). Anti-His tag monoclonal antibody was from Novagen (San Diego, CA). The Phototope-HRP Western Blot Detection System was from Cell Signaling (Beverley, MA). Rabbit polyclonal anti-P450 3A37 serum was raised against the peptide sequence “SQKSDSDGKNSHKA” (amino acids 278-291) by Genemed Synthesis (San Antonio, TX). This peptide sequence was selected by the manufacturer while taking into consideration that it should be antigenic, hydrophilic, and readily accessible. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Rapid Amplification of cDNA Ends (RACE). P450 3A37 cDNA was amplified from 1-day-old male turkey liver by 3′-5′RACE procedures described previously (15, 21). Briefly, small segments of RNA later storing turkey liver were homogenized, and RNA was isolated using Poly (A) Pure mRNA purification kit (Ambion, Austin, TX). Isolated RNA was further used as the RACE template. The RACE procedure as mentioned in BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto, CA) was followed. Primers used in 3′-RACE were designed upon the basis of the sequence alignment of P450 3A4 in human and cattle, 3A4V2 in Cercopithecus aethiops (African green monkey), 3A44 in mouse, 3A66 in Macaca mulatta (rhesus monkey), and 3A45 in Onchorhynchus mykiss (rainbow trout) using ClustalW (www.ebi.ac.uk). 5′-RACE primers were designed on the basis of sequences of 3′-RACE as well as the sequence alignments. Products of the PCR were cloned into the pCR 4-TOPO vector and sequenced at the core facility at Utah State University.

Sequences were analyzed by Lasergene SeqMan II software (DNASTAR Inc., Madison, WI). Construction of Expression Plasmid and Bacterial Expression. An E. coli based recombinant P450 system was used for P450 3A4 homologue identification (15). Expression vector construction is as described previously (21). We chose the pCW vector, developed by Dahlquist (22), which contains two tac promoters (inducible by isopropyl-β-D-thiogalactopyranoside: IPTG) cassettes upstream of an NdeI (CATATG) restriction enzyme cloning site coincident with the initiation ATG codon. This vector contains a strong transcription termination mechanism and a phage M13 origin of DNA replication. Transcription prior to the addition of inducing agents is inhibited by the LacI gene encoding a lac repressor molecule. The original bicistronic pCW1A2/hNPR vector developed by Dr. F. Peter Guengerich was generously provided by Dr. Gary Yost, University of Utah. Bicistronic expression constructs consisting of the coding sequence of P450 3A37 followed by human NADPH-P450 reductase (hNPR), the redox partner to P450, were constructed as described (15, 21). P450s are difficult to express in their native form due to the hydrophobic amino acid rich N-terminal region; therefore, a truncated version of the gene was constructed to enhance its expression in E. coli (23) (Figure 1). Specifically, 11 amino acids were deleted in the N-terminus, and the first 8 codons had a modified nucleotide sequence to enhance its expression. A 6X-His tag was added in the C-terminal region to facilitate purification (24). The original pCW1A2/hNPR vector was digested with NdeI and XbaI to excise the P450 1A2 coding sequence for the construction of expression constructs. Clones of P450 3A37 digested with NdeI and XbaI were ligated with the gel-purified vector fragment and PCR-amplified using T4 DNA ligase. This ligation mix was transformed into DH5RF′IQ cells. Heterologous expression of P450 3A37 in E. coli was performed (25). A single colony of DH5RF′IQ E. coli transformants was inoculated in 5 mL of Luria-Bertani ampicillin medium. It was then allowed to grow overnight at 37 °C with vigorous shaking. Four hundred fifty milliliters of modified Terrific-Broth ampicillin medium with trace elements was then used for further inoculation of the starter culture at 1:100 dilutions. Cultures were grown at 37 °C to an OD600 of 0.3-0.5. After an equilibration step of 30 min at 30 °C, induction was initiated by adding 0.5 mM δ-aminolevulinic acid and 1 mM IPTG. Cells were incubated at 30 °C with shaking (150 rpm) and harvested after 23 h, and levels of P450 expression was determined by a reduced CO/reduced difference spectrum (26) (Spectronic GENESYS 6 UV-vis spectrophotometer, Thermo Spectronic, Rochester, NY). E. coli membranes and cytosol were prepared as described (15). All kinetic assays were performed with P450 3A37 E. coli membranes.

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Figure 2. Complete cDNA and predicted amino acid sequence of turkey P450 3A37, with 5′- and 3′-UTRs. ORF, 1512bp encoding 504 aa; 5′UTR, 53 bp; 3′-UTR, 384 bp; total cDNA length, 1949 bp. Start and stop codons are bold faced, and the heme-binding motif, usually represented as FXXGXXXCXG, is underlined.

Table 1. Nucleic Acid and Amino Acid Sequence Identity and Similarity between Turkey P450 3A37 and Other P450 3As (EMBOSS-Align) nucleic acid

amino acid

cytochrome P450

species

% identity

% identity

% similaritya

P450 3A37 P450 3A P450 3A13 P450 3A13 P450 3A4 P450 3A5

chicken Xenopus rat mouse human human

93 64 67 67 66 66

92 56 62 60 58 58

97 75 79 79 76 76

a Percentage similarity of the amino acid sequence is determined on the basis of the EBLOSUM62 matrix.

Immunodetection of Recombinant P450 3A37. E. coli membranes expressing P450 3A37, control vector (pCWOri+ without P450 and reductase inserts), and turkey liver microsomes were run on a 4-15% Tris-HCl Ready Gel (Bio-Rad). Immunoblotting was performed according to the supplier’s instructions (Qiagen, Valen-

cia, CA). To reduce the extraneous bands caused by the recognition of bacterial proteins, the rabbit polyclonal anti-P450 3A37 serum was first preadsorbed by pCWOri+ E. coli membranes (15, 27). Briefly, 120 µL of pCWOri+ membranes (22 mg of protein) was added to 2 mL of antiserum (111 mg protein) and incubated at 4 °C for 2.5 h for preadsorption. The samples were centrifuged at 13000g for 10 min at 4 °C, and supernatants were used as the primary antibody (1:4000). HRP-conjugated goat antirabbit IgG (1: 5000) were used as the secondary antibody. Chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce, Rockford, IL) was used for signal detection, and images were captured using Nucleovision E20 Imaging Workstation (Nucleotech, Hayward, CA). Quantification of AFB1 Metabolites. The exo-AFBO intermediate is unstable and has a short half-life; therefore, it was determined indirectly as a stable, trapped AFBO-GSH conjugate using conditions we have described previously (15). A reaction mixture consisting of E. coli membranes expressing P450 3A37 (0.709 µM), butylated hydroxyanisole (BHA)-induced mouse cytosol (∼800 µg) as a source of GST, 2 mM NADPH, 5 mM GSH, and 0.1-1000



Figure 3. (A) Immunoblot showing 60 kDa bands in both turkey liver microsomes and in P450 3A37-expressing E. coli membranes. A minor band which was detected at around 80 kDa was presumably a bacterial protein, present in E. coli membranes of P450 3A37 and in those of pCWOri+ (the empty vector control). CO-difference spectra of (B) whole cells and (C) membranes.

µM AFB1 in spectral grade dimethyl sulfoxide (DMSO) was made in epoxide trapping buffer (5 mM MgCl2, 25 mM KCl, 0.25 mM sucrose, and 80 mM potassium phosphate, pH 7.6) to give a final volume of 250 µL. The reactions were incubated at 37 °C for 20 min with gentle shaking and stopped by adding 250 µL of cold methanol spiked with 24 µM aflatoxin G1 (AFG1) as an internal standard. Inhibitor or anti-P450 3A37 antibodies whenever used were incubated with the membranes for 10 min in an ice-bath before adding other components of the reaction. The P450 3A4 specific inhibitor 17R-ethynylestradiol was used as described previously (40). This treatment was done to provide extra time for the inhibitor or the antibody to bind to the enzyme. The samples were kept overnight at -20 °C to facilitate protein precipitation and then were centrifuged at 13000g for 10 min. The supernatant was filtered through a 0.2 µm nylon membrane before 100 µL was injected into the HPLC. Metabolites were separated on a Shimadzu LC system (Shimadzu, Pleasanton, CA), equipped with a model LC20AD pump, a model SPD-20AV UV-vis detector, and an Econosphere C18 (150 mm × 4.6 mm) column (Alltech Associates, Deerfield, IL), which was kept at 40 °C. The elution of the peaks was monitored by UV absorbance (λ ) 254 nm). The mobile phases used in this assay were as described previously (10). Amounts of metabolite formation were calculated by establishing calibration curves between the peak areas in the chromatograms and the amounts of metabolites injected, using authentic AFBO-GSH and AFQ1 standards. Nifedipine Oxidation Activity. Nifedipine oxidation was determined as described previously (28). Inhibitors or anti-P450 3A37 antibodies were tested on this activity in a similar way, as described above.

Data Analysis. Kinetic data were analyzed by SigmaPlot Enzyme Kinetics Module software (Systat, San Jose, CA). Data for exoAFBO and AFQ1 formation were analyzed by nonlinear regression using the general allosteric model, the Hill equation to calculate Vmax and S50 [V ) Vmax [S]n/ ([S50]n + [S]n)]. In this equation, n (Hill coefficient) represents the degree of cooperativity.

Results RACE. Sequence analysis revealed that this gene has an ORF of 1512 bp coding for 504 amino acids. In addition, it had 53 bp in the 5′-untranslated region (UTR) and 384 bp in the 3′UTR with a total of 1949 bp (Figure 2). Predicted molecular mass of this P450 3A4 homologue is 58 kDa. The heme-binding motif, usually represented as FXXGXXXCXG, was identified in P450 3A37 as FGAGPRNCIG (Figure 2). Sequence identity comparison of this gene and the P450 3A class gene in other species is shown in Table 1. These comparisons which were made using the EMBOSS-Align program (www.ebi.ac.uk) revealed that this gene has 93% and 92% nucleic acid and amino acid identity, respectively, to the chicken P450 3A37 (29) and was thus assigned as P450 3A37 by Dr. David Nelson of the University of Tennessee. Bacterial Expression and Immunodetection. As frequently observed with unmodified P450s (30), the wild-type P450 3A37 did not express detectable P450 activity as determined by the reduced CO/reduced difference spectrum (26). The truncated tP450 3A37 expressed 250-400 nmol of 3A37 per liter of the

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Figure 4. V vs S plot of exo-AFBO and AFQ1 formation. Kinetics of AFB1 oxidation by E. coli expressed P450 3A37. A sigmoidal relationship between substrate concentration and product formation was observed, suggesting that exo-AFBO and AFQ1 formation is driven by an allosteric interaction between P450 3A37 and AFB1, showing positive cooperativity. The data was analyzed by Hill’s equation.

Figure 5. Nifedipine oxidation activity of E. coli membranes expressing P450 3A37. Activity followed Michaelis-Menten kinetics with the following parameters: Vmax ) 3.4 ( 0.25 nmol min/nmol/P450 and Km ) 98 ( 16 µM (R2 ) 0.84). Points are means ((SE) (n ) 3).

Table 2. Hill Equation Kinetic Constants for P450 3A37 Catalyzed AFB1 Oxidation in E. coli Expressed P450 3A37, at AFB1 Concentrations of 0.1-1000 µMa metabolite

Vmax ( SEb

Km ( SEb

n ( SEb

R2

exo-AFBO AFQ1

1.45 ( 0.07 7.86 ( 0.75

287 ( 21 302 ( 51

1.9 ( 0.18 1.6 ( 0.26

0.98 0.96

a n is the degree of cooperativity. Vmax and Km measured as nmol/ min/nmol P450 and µM, respectively. b Data from three replicates.

culture, as determined by the CO difference spectra using whole cells (Figure 3). P450 3A37 was recovered in both the membrane and cytosol: approximately 26% and 25-45%, respectively. E. coli membranes expressing recombinant P450 3A37 also expressed active hNPR. The activity of hNPR, as measured by the reduction of cytochrome c, was 0.54 µmol/ min/mg total protein. As compared to the activity of purified hNPR expressed in E. coli from the same plasmid (50 µmol/ min/mg total protein), we determined that hNPR comprised 2.8% (w/w) of the proteins in tP450 3A37 E. coli membrane (vs 8% for P450), which corresponded to a hNPR-to-P450 molar ratio of about 1:4. Polyclonal anti-P450 3A37 antibodies detected a band of about 58 kDa in E. coli expressed P450 3A37 and in turkey liver microsomes (Figure 3). AFB1 Metabolism and Nifedipine Oxidation Activity. E. coli expressed P450 3A37 oxidized AFB1 to exo-AFBO and AFQ1, which when plotted V versus S, exhibited a sigmoidal relationship, suggesting that the formation of both products is driven by an allosteric interaction between AFB1 and P450 3A37, showing positive cooperativity (Figure 4). The data was analyzed by the general allosteric model using the Hill equation. The Hill coefficient (n) value for the formation of exo-AFBO and AFQ1 was 1.9 and 1.6, respectively (Table 2). Like human P450 3A4 (31), 3A37 possessed nifedipine oxidation activity which, unlike AFB1 metabolism, conformed to simple Michaelis-Menten kinetics. The Km and Vmax for this activity were 98 ( 16 µM and 3.4 ( 0.25 nmol/min/ nmol P450 (R2 ) 0.96), respectively (Figure 5). Inhibition by Specific P450 Inhibitors and Anti-P450 3A37 Antibodies. The effects of specific inhibitors to mammalian P450s on exo-AFBO formation as well as on nifedipine oxidation activity were then examined. The specific P450 3A4 inhibitor 17R-ethynylestradiol was the most effective of those tested (IC50 ) 40 µM) (Figure 6).

Figure 6. Inhibition of AFB1 (100 µM) oxidation to exo-AFBO by E. coli expressed P450 3A37. (A) P450 3A4 prototype inhibitor 17Rethynylestradiol inhibited the formation of exo-AFBO (IC50 ) 40 µM). Erythromycin was less efficient. (B) Inhibitors to other mammalian P450s were either slightly effective or not effective. Points are means (n ) 3).

Erythromycin, a P450 3A1/4 inhibitor, was moderately effective. Inhibitors to other P450s were either slightly effective or not effective (Figure 6). At the same concentrations, 17R-ethynylestradiol also inhibited nifedipine oxidation activity (IC50 ) 74 µM) (Figure 7). Anti-P450 3A37 antibodies inhibited the formation of both exo-AFBO and nifedipine oxidation activity, as shown in Figure 8.


Figure 7. 17R-Ethynylestradiol, a specific inhibitor to P450 3A4, inhibited nifedipine oxidation activity of E. coli expressed P450 3A37 (IC50 ) 74 µM). Erythromycin was less effective. Points are means (n ) 3).

Figure 8. Polyclonal anti-P450 3A37 antibody (5 µg/mL/nmol P450) inhibited AFB1 epoxidation and nifedipine oxidation activity of E. coli expressed P450 3A37. Points are means (n ) 3).

Discussion Many P450s bioactivate xenobiotics such as AFB1, converting them into highly reactive electrophilic intermediates, which cause mutation and carcinogenesis. Although AFB1 is metabolized by P450s to endo- and/or exo-AFBO, it is the exostereoisomer which binds to DNA, RNA, and other cellular macromolecules to elicit the toxic responses to this mycotoxin (32). The extreme sensitivity of turkeys to AFB1 is due, at least in part, to the presence of hepatic P450s with high epoxidation activity. In turkey liver microsomes, both P450 1A and 3A homologues are responsible for the metabolism of AFB1 to the toxic and carcinogenic exo-AFBO, as seen in humans and other animals (10, 15, 21). Here, we report that the turkey liver possesses P450 3A37, a homologue to human P450 3A4 with AFB1 epoxidation and nifedipine oxidation activity. The nucleic acid sequence of this gene was remarkably close to chicken P450 3A37 reported by Ourlin et al. (29), with 93% identity. While the sequence of this gene was 66% identical to human P450 3A4, the catalytic activity of the E. coli expressed P450 3A37 closely resembled that of its human counterpart. Turkey P450 3A37 possessed activity toward the prototypical P450 3A4 substrate nifedipine (31) and also oxidized AFB1 in a manner similar to that of human P450 3A4 (5). Inhibition of the catalytic activities of P450 3A37 by 17R-ethynylestradiol further supports its designation as a P450 3A4 homologue.


Generally, P450s require alterations in the hydrophobic N-terminal nucleotide sequence (5′-end) to facilitate translation by E. coli (23). The N-terminal truncated sequence of wildtype P450 3A37 was produced by deleting 11 amino acids and modifying the nucleotide sequence of the first 8 codons to facilitate translation by E. coli. These modifications are reported to reduce the free energy for base pairing while inhibiting the formation of secondary structures by mRNA, which could inhibit translation (2). Subsequently, truncated P450 3A37 expressed in E. coli produced substantial amounts of active P450, which was confirmed by immunoblots showing a band of apparent molecular mass of about 58 kDa. The addition of hNPR, in our bicistronic constructs ensured that the electron transfer to P450 was not rate limiting: the hNPR-to-P450 ratio in the E. coli membranes was 1:4, similar to what we previously reported for turkey P450 1A5 (15) and greater than the previously reported ratios of 1:5-20 in mammalian liver microsomes (1, 33, 34). Striking similarities between the catalytic activities of E. coli expressed turkey P450 3A37 and human P450 3A4 were observed. For example, turkey P450 3A37 efficiently metabolized AFB1 to exo-AFBO and AFQ1 and also possessed nifedipine oxidation activity. Furthermore, these catalytic activities were completely inhibited by P450 3A4 specific inhibitor 17R-ethynylestradiol. It must be emphasized that the P450 prototypical substrates and inhibitors used in this study were developed for mammalian P450s, and the specificities of these compounds for avian P450s have not been rigorously validated. Erythromycin, an inhibitor to P450 3A1/4, also inhibited the formation of exo-AFBO as well as nifedipine oxidation activity but at concentrations higher than that observed for 17R-ethynylestradiol (g800 µM). While 17R-ethynylestradiol efficiently inhibited the catalytic activities of P450 3A37, erythromycin was moderately effective. The IC50 values for the inhibition of P450 3A4 for the metabolism of laquinimod by 17R-ethynylestradiol and erythromycin are reported as 235 and 24 µM, respectively (36). While this IC50 value for 17R-ethynylestradiol is higher than that of P450 3A37, those for erythromycin are much lower. Such interspecies differences are to be expected, but these observations may also be explained by the fact that the reported values are for different substrates. That metabolic specificity of P450 3A37 confirms our previous observation of the involvement of a P450 3A4 homologue (along with a P450 1A2 homologue) in AFB1 bioactivation in turkey liver microsomes (10, 15). Similar to P450 1A5, turkey P450 3A37 resembled its human counterpart with respect to the metabolite profile and to the kinetics of AFB1 oxidation. Like human P450 3A4, 3A37 catalysis of AFB1 is driven by an allosteric interaction, showing positive cooperativity. Human P450 3A4 is reported to have a large and conformationally dynamic active site which can accommodate multiple substrates or effector molecules, the binding of which leads to allosteric kinetic behavior in that substrate binding to P450 3A4 facilitates the binding of additional molecules of substrate, enhancing turnover (35). Similar to human P450 3A4 (5), the n value in the Hill plot for P450 3A37 for AFB1 oxidation was approximately two. The Km values determined for P450 3A37 were almost 2-fold greater than those for human P450 3A4 (302 µM vs 139 µM for AFQ1; 287 µM for exoAFBO in our study vs 133 µM for AFBO in ref 5), though this comparison is not accurate since the authors (5) did not distinguish the endo- and exo- isomers. The Vmax value of AFQ1 formation was almost 8-fold lower for P450 3A37 than that for human P450 3A4 (7.86 vs 61 nmol/min/nmol P450), while that

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of exo-AFBO formation in our study was more than 4-fold lower than that of the AFBO formation measured for human P450 3A4 (1.45 vs 6.2 nmol/min/nmol P450) (5). Such kinetic differences could be explained by interspecies variations of P450s but is more likely due to the different in Vitro systems used to model enzyme activity. Observed differences in the activities of P450 3A4 and 3A37 may be attributable to sequence differences in the substrate recognition sites (SRS). Although the SRSs were not totally conserved in P450 3A37, several key residues in the SRSs shown to be critical in the catalytic activities of P450 3A4 were conserved in 3A37. These included Cys 98, important in the interaction of 3A4 with the NADPH cytochrome P450 reductase (37); Ser 119, critical in the catalysis and regioselectivity of 3A4 for the substrates (38); Leu 210, plays a role in the cooperativity and regioselectivity of 3A4 for AFB1 (39); and Phe 304, important in AFB1 binding at the active site leading to the formation of AFQ1 or exo-AFBO (39). Asn 206, which plays an important role in the cooperativity and the regioselectivity of 3A4 for AFB1, was not conserved in 3A37 and was replaced with Glu. Although P450 3A37 and turkey P450 1A5 (15) exhibited different kinetics, a comparison of the kinetic constants of exoAFBO formation suggests that the former may be more efficient. The Vmax of P450 3A37 is more than two times that of P450 1A5 (1.45 ( 0.07 vs 0.61 ( 0.04 nmol/min/nmol P450). However, it must be emphasized that the Km values of P450 3A37 for AFB1 is almost 4-fold higher than that of P450 1A5 (287 ( 21 vs 65 ( 12 µM). The catalytic efficiencies of P450 3A37 (0.005 min-1 µM-1) vs 1A5 (0.009 min-1 µM-1) indicates 1A5 as more efficient than 3A37. We have previously reported that the extreme sensitivity of turkeys to AFB1 is due, in part, to a combination of efficient P450 mediated epoxidation and deficient detoxification by GSTs. While turkeys possess several hepatic GSTs (41), none thus far have been identified which are capable of conjugating the epoxide with the endogenous glutathione (10). In conclusion, P450 3A37 is important in mediating the toxicity of AFB1 in turkeys. This gene was heterologously expressed in E. coli, in quantities sufficient to support the catalytic activities, which were similar to those of the human P450 3A4. Because of the role of human P450 3A4 in AFB1 oxidation to exo-AFBO (5, 19, 20), we believe that the identification of this homologue from turkeys is important in discerning the mechanisms of extreme sensitivity of this species to AFB1. Of particular importance was the metabolism of AFB1 by the E. coli expressed protein to exoAFBO and AFQ1. exo-AFBO, a highly reactive electrophilic intermediate, is responsible for carcinogenic and mutagenic effects of AFB1. Thus, we conclude that P450 3A37, along with P450 1A5, plays an important role in the extreme sensitivity of turkeys to AFB1. Acknowledgment. We thank Dr. Lynn Bagley of the Moroni Feed Cooperative for generously providing turkeys and feed for this study. This research was supported in part by competitive grant 2007-35205-17880 from the USDA National Institute of Food and Agriculture Animal Genome program, and from the Utah Agricultural Experiment Station.

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Cloning, Expression and Functional Characterization of Cytochrome

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