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Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B 1

View Article: PubMed Central - PubMed

ABSTRACT

Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.

No MeSH data available.


Purification, CO difference spectra, and AFB1 oxidation activity of recombinant pig CYP3A29. (A) Expression and purification of CYP3A29 in E. coli DH5α. The band around 60 kDa is the protein of interest (arrow). M: protein marker; Lane 1: non-IPTG induced total cell lysates; Lane 2: IPTG-induced total cell lysates; Lane 3: the solubilized membrane components; Lane 4: purified recombinant pig CYP3A29. (B) Fe2+ ∙ CO vs. Fe2+ difference spectra of CYP3A29 and the mutants. The spectra were recorded at 25 °C and in 100 mM Tris∙HCl buffer (pH 7.4) containing 10 mM CHAPS, 20% glycerol, and 1 mM EDTA. (C) Top: the HPLC chromatogram of AFB1 metabolized by quail liver microsomes; Middle: HPLC chromatogram of AFB1 metabolized by recombinant CYP3A29; Bottom: CYP3A29 was inhibited by 5 μM ketoconazole and no metabolite formed. The dihydrotriol-tris is an indirect indication of the formation of the major metabolite AFBO and AFG1 was added into the reaction mixture as an internal standard. HPLC eluent was monitored by fluorescence (λexcitation = 365 nm, λemission = 440 nm).
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toxins-08-00267-f001: Purification, CO difference spectra, and AFB1 oxidation activity of recombinant pig CYP3A29. (A) Expression and purification of CYP3A29 in E. coli DH5α. The band around 60 kDa is the protein of interest (arrow). M: protein marker; Lane 1: non-IPTG induced total cell lysates; Lane 2: IPTG-induced total cell lysates; Lane 3: the solubilized membrane components; Lane 4: purified recombinant pig CYP3A29. (B) Fe2+ ∙ CO vs. Fe2+ difference spectra of CYP3A29 and the mutants. The spectra were recorded at 25 °C and in 100 mM Tris∙HCl buffer (pH 7.4) containing 10 mM CHAPS, 20% glycerol, and 1 mM EDTA. (C) Top: the HPLC chromatogram of AFB1 metabolized by quail liver microsomes; Middle: HPLC chromatogram of AFB1 metabolized by recombinant CYP3A29; Bottom: CYP3A29 was inhibited by 5 μM ketoconazole and no metabolite formed. The dihydrotriol-tris is an indirect indication of the formation of the major metabolite AFBO and AFG1 was added into the reaction mixture as an internal standard. HPLC eluent was monitored by fluorescence (λexcitation = 365 nm, λemission = 440 nm).

Mentions: The recombinant pig CYP3A29 and its mutants were cloned and expressed as His6-tagged fusion protein in Escherichia coli. CYP3A29 WT with an apparent molecular mass of 60 kDa was obtained by affinity chromatography, and its purity exceeded 95% (Figure 1A, lane 4). The production of protein was estimated to be approximately 20.8 mg/L bacterial culture. Fe2+ ∙ CO vs. Fe2+ difference spectra of CYP3A29 and the mutants are shown in Figure 1B. The recombinant proteins had a strong absorbance around 450 nm and no absorbance peak at 420 nm, indicating that CYP3A29 WT and the mutants expressed in the E. coli membrane possessed the spectral characteristics of functional P450.


Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B 1
Purification, CO difference spectra, and AFB1 oxidation activity of recombinant pig CYP3A29. (A) Expression and purification of CYP3A29 in E. coli DH5α. The band around 60 kDa is the protein of interest (arrow). M: protein marker; Lane 1: non-IPTG induced total cell lysates; Lane 2: IPTG-induced total cell lysates; Lane 3: the solubilized membrane components; Lane 4: purified recombinant pig CYP3A29. (B) Fe2+ ∙ CO vs. Fe2+ difference spectra of CYP3A29 and the mutants. The spectra were recorded at 25 °C and in 100 mM Tris∙HCl buffer (pH 7.4) containing 10 mM CHAPS, 20% glycerol, and 1 mM EDTA. (C) Top: the HPLC chromatogram of AFB1 metabolized by quail liver microsomes; Middle: HPLC chromatogram of AFB1 metabolized by recombinant CYP3A29; Bottom: CYP3A29 was inhibited by 5 μM ketoconazole and no metabolite formed. The dihydrotriol-tris is an indirect indication of the formation of the major metabolite AFBO and AFG1 was added into the reaction mixture as an internal standard. HPLC eluent was monitored by fluorescence (λexcitation = 365 nm, λemission = 440 nm).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037493&req=5

toxins-08-00267-f001: Purification, CO difference spectra, and AFB1 oxidation activity of recombinant pig CYP3A29. (A) Expression and purification of CYP3A29 in E. coli DH5α. The band around 60 kDa is the protein of interest (arrow). M: protein marker; Lane 1: non-IPTG induced total cell lysates; Lane 2: IPTG-induced total cell lysates; Lane 3: the solubilized membrane components; Lane 4: purified recombinant pig CYP3A29. (B) Fe2+ ∙ CO vs. Fe2+ difference spectra of CYP3A29 and the mutants. The spectra were recorded at 25 °C and in 100 mM Tris∙HCl buffer (pH 7.4) containing 10 mM CHAPS, 20% glycerol, and 1 mM EDTA. (C) Top: the HPLC chromatogram of AFB1 metabolized by quail liver microsomes; Middle: HPLC chromatogram of AFB1 metabolized by recombinant CYP3A29; Bottom: CYP3A29 was inhibited by 5 μM ketoconazole and no metabolite formed. The dihydrotriol-tris is an indirect indication of the formation of the major metabolite AFBO and AFG1 was added into the reaction mixture as an internal standard. HPLC eluent was monitored by fluorescence (λexcitation = 365 nm, λemission = 440 nm).
Mentions: The recombinant pig CYP3A29 and its mutants were cloned and expressed as His6-tagged fusion protein in Escherichia coli. CYP3A29 WT with an apparent molecular mass of 60 kDa was obtained by affinity chromatography, and its purity exceeded 95% (Figure 1A, lane 4). The production of protein was estimated to be approximately 20.8 mg/L bacterial culture. Fe2+ ∙ CO vs. Fe2+ difference spectra of CYP3A29 and the mutants are shown in Figure 1B. The recombinant proteins had a strong absorbance around 450 nm and no absorbance peak at 420 nm, indicating that CYP3A29 WT and the mutants expressed in the E. coli membrane possessed the spectral characteristics of functional P450.

View Article: PubMed Central - PubMed

ABSTRACT

Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.

No MeSH data available.