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

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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.


AFB1 oxidation by CYP3A29 and the mutants. (A–D) HPLC chromatograms of AFB1 metabolized by WT, S119A, F304A, and T309A, respectively; (E) histograms of relative activities of CYP3A29 and the mutants towards AFB1. The dihydrotriol–tris is an indirect indication of the formation of the major metabolite AFBO and AFG1 was added as an internal standard. HPLC eluent was monitored by fluorescence (λexcitation = 365 nm, λemission = 440 nm). ** p < 0.01, Values are means ± SE (n = 3).
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toxins-08-00267-f003: AFB1 oxidation by CYP3A29 and the mutants. (A–D) HPLC chromatograms of AFB1 metabolized by WT, S119A, F304A, and T309A, respectively; (E) histograms of relative activities of CYP3A29 and the mutants towards AFB1. The dihydrotriol–tris is an indirect indication of the formation of the major metabolite AFBO and AFG1 was added as an internal standard. HPLC eluent was monitored by fluorescence (λexcitation = 365 nm, λemission = 440 nm). ** p < 0.01, Values are means ± SE (n = 3).

Mentions: To explore the regioselectivity and specificity of pig CYP3A29 towards AFB1, we carried out the incubation of CYP3A29 and its mutants with AFB1. Oxidizing activities of all recombinant enzymes towards AFB1 can be relatively quantified and compared by analyzing the ratio of peak area of the main product to that of AFG1 (the original data was shown in Table S1). The relative activities of WT and its mutants can be seen in Figure 3E. Compared with WT (Figure 3A), the substitution of Ser119 with Ala resulted in the middle impairment (about 50%) of AFB1 exo-epoxidation (Figure 3B). Unexpectedly, replacement of Phe304 with Ala led to over 2-fold increase of AFB1 epoxidation activity (Figure 3C), which is totally opposite to the result observed in human CYP3A4 [36]. For Thr309, its substitution with Ala resulted in the significant impairment (about 80%) of AFB1 exo-epoxidation (Figure 3D), which implied that threonine at the position 309 may play an important role in the activation of AFB1.


Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B 1
AFB1 oxidation by CYP3A29 and the mutants. (A–D) HPLC chromatograms of AFB1 metabolized by WT, S119A, F304A, and T309A, respectively; (E) histograms of relative activities of CYP3A29 and the mutants towards AFB1. The dihydrotriol–tris is an indirect indication of the formation of the major metabolite AFBO and AFG1 was added as an internal standard. HPLC eluent was monitored by fluorescence (λexcitation = 365 nm, λemission = 440 nm). ** p < 0.01, Values are means ± SE (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

toxins-08-00267-f003: AFB1 oxidation by CYP3A29 and the mutants. (A–D) HPLC chromatograms of AFB1 metabolized by WT, S119A, F304A, and T309A, respectively; (E) histograms of relative activities of CYP3A29 and the mutants towards AFB1. The dihydrotriol–tris is an indirect indication of the formation of the major metabolite AFBO and AFG1 was added as an internal standard. HPLC eluent was monitored by fluorescence (λexcitation = 365 nm, λemission = 440 nm). ** p < 0.01, Values are means ± SE (n = 3).
Mentions: To explore the regioselectivity and specificity of pig CYP3A29 towards AFB1, we carried out the incubation of CYP3A29 and its mutants with AFB1. Oxidizing activities of all recombinant enzymes towards AFB1 can be relatively quantified and compared by analyzing the ratio of peak area of the main product to that of AFG1 (the original data was shown in Table S1). The relative activities of WT and its mutants can be seen in Figure 3E. Compared with WT (Figure 3A), the substitution of Ser119 with Ala resulted in the middle impairment (about 50%) of AFB1 exo-epoxidation (Figure 3B). Unexpectedly, replacement of Phe304 with Ala led to over 2-fold increase of AFB1 epoxidation activity (Figure 3C), which is totally opposite to the result observed in human CYP3A4 [36]. For Thr309, its substitution with Ala resulted in the significant impairment (about 80%) of AFB1 exo-epoxidation (Figure 3D), which implied that threonine at the position 309 may play an important role in the activation of AFB1.

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&nbsp;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.