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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.


Molecular models of AFB1 docked into CYP3A29 and the mutants. (A) The docking conformation of AFB1 in the active site of CYP3A29; (B) the docking conformation of AFB1 in F304A. The best conformation of AFB1 was docked into F304A (shown as thick sticks), while that of AFB1 in WT is shown as thin sticks; (C) The docking conformation of AFB1 in T309A. CYP3A29 is shown in a ribbon format, and an iron ion is shown as a sphere. The examined amino acid residues are green, and the heme is orange. Color scheme: red for oxygen atoms and blue for nitrogen atoms. Hydrogen bonds are shown as yellow dashed lines, and distances between atoms are shown with black dashed lines and given in angstroms. Residues 368–371 and 476–484 are not shown, for clarity. The figures are rendered with PyMOL (Version 1.8, Schrodinger, LLC., New York, NY, USA, 2015) [42].
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toxins-08-00267-f004: Molecular models of AFB1 docked into CYP3A29 and the mutants. (A) The docking conformation of AFB1 in the active site of CYP3A29; (B) the docking conformation of AFB1 in F304A. The best conformation of AFB1 was docked into F304A (shown as thick sticks), while that of AFB1 in WT is shown as thin sticks; (C) The docking conformation of AFB1 in T309A. CYP3A29 is shown in a ribbon format, and an iron ion is shown as a sphere. The examined amino acid residues are green, and the heme is orange. Color scheme: red for oxygen atoms and blue for nitrogen atoms. Hydrogen bonds are shown as yellow dashed lines, and distances between atoms are shown with black dashed lines and given in angstroms. Residues 368–371 and 476–484 are not shown, for clarity. The figures are rendered with PyMOL (Version 1.8, Schrodinger, LLC., New York, NY, USA, 2015) [42].

Mentions: To establish a binding model and understand the details of interaction between ligand and receptor, we carried out the molecular docking study on AFB1 with CYP3A29 and its mutants. One hundred docking conformations generated by AutoDock 4.2 were clusters with an RMSD of 2.0 Å and ranked by the lowest docked energy. Two clusters were produced by the docking simulations when AFB1 was docked into receptor CYP3A29 WT, F304A, and S119A respectively, and only one for T309A. For WT, the first cluster contained 37% conformers and had −8.58 kcal/mol of the lowest binding energy (Table 2). It can be seen from the side view of the docking model that AFB1 was located to the upper right of the heme iron (Figure S2), in which orientation the epoxy bond could form only on the left of the terminal furan ring. Stereochemically, this is AFB1-exo-8,9-epoxide. Consistent with the previous reports [31,36], the docking only produced the orientation that was favorable for forming exo-8,9-epoxidation while no orientation for endo-8,9-epoxidation. In the first cluster, AFB1 almost stood vertically onto the heme, the redox center, with its C8 oriented towards the heme iron (Figure 4A). Furthermore, the measured interatomic distance between C8 and heme iron was 2.7 Å (Figure 4A), which allowed the contact of the ferric peroxy anion and led to the electrons’ transfer from the substrate and the subsequent formation of AFBO. Therefore, the conformations in this cluster predicted formation of active metabolite. For the other cluster, on the contrary, the docking result showed that the oxygen atom at C1 of AFB1 was oriented towards the heme iron, whereas C8, C9-double bond was far away from the oxidized center, in which case no AFBO could be formed, thus these conformations were thought inactive. It also can be seen from the putative binding model that AFB1 interacted with residues in the active site by forming two hydrogen bonds: one was between side chain hydroxyl group of Tyr309 and O6 or O7 of AFB1; the other was between the oxygen atom at C4 of AFB1 and the hydrogen in a peptide bond formed by Lys212 with Phe213 (Figure 4A). It is speculated that these two hydrogen bonds ought to play special roles in maintaining the stable binding of AFB1 in the active center.


Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B 1
Molecular models of AFB1 docked into CYP3A29 and the mutants. (A) The docking conformation of AFB1 in the active site of CYP3A29; (B) the docking conformation of AFB1 in F304A. The best conformation of AFB1 was docked into F304A (shown as thick sticks), while that of AFB1 in WT is shown as thin sticks; (C) The docking conformation of AFB1 in T309A. CYP3A29 is shown in a ribbon format, and an iron ion is shown as a sphere. The examined amino acid residues are green, and the heme is orange. Color scheme: red for oxygen atoms and blue for nitrogen atoms. Hydrogen bonds are shown as yellow dashed lines, and distances between atoms are shown with black dashed lines and given in angstroms. Residues 368–371 and 476–484 are not shown, for clarity. The figures are rendered with PyMOL (Version 1.8, Schrodinger, LLC., New York, NY, USA, 2015) [42].
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Related In: Results  -  Collection

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toxins-08-00267-f004: Molecular models of AFB1 docked into CYP3A29 and the mutants. (A) The docking conformation of AFB1 in the active site of CYP3A29; (B) the docking conformation of AFB1 in F304A. The best conformation of AFB1 was docked into F304A (shown as thick sticks), while that of AFB1 in WT is shown as thin sticks; (C) The docking conformation of AFB1 in T309A. CYP3A29 is shown in a ribbon format, and an iron ion is shown as a sphere. The examined amino acid residues are green, and the heme is orange. Color scheme: red for oxygen atoms and blue for nitrogen atoms. Hydrogen bonds are shown as yellow dashed lines, and distances between atoms are shown with black dashed lines and given in angstroms. Residues 368–371 and 476–484 are not shown, for clarity. The figures are rendered with PyMOL (Version 1.8, Schrodinger, LLC., New York, NY, USA, 2015) [42].
Mentions: To establish a binding model and understand the details of interaction between ligand and receptor, we carried out the molecular docking study on AFB1 with CYP3A29 and its mutants. One hundred docking conformations generated by AutoDock 4.2 were clusters with an RMSD of 2.0 Å and ranked by the lowest docked energy. Two clusters were produced by the docking simulations when AFB1 was docked into receptor CYP3A29 WT, F304A, and S119A respectively, and only one for T309A. For WT, the first cluster contained 37% conformers and had −8.58 kcal/mol of the lowest binding energy (Table 2). It can be seen from the side view of the docking model that AFB1 was located to the upper right of the heme iron (Figure S2), in which orientation the epoxy bond could form only on the left of the terminal furan ring. Stereochemically, this is AFB1-exo-8,9-epoxide. Consistent with the previous reports [31,36], the docking only produced the orientation that was favorable for forming exo-8,9-epoxidation while no orientation for endo-8,9-epoxidation. In the first cluster, AFB1 almost stood vertically onto the heme, the redox center, with its C8 oriented towards the heme iron (Figure 4A). Furthermore, the measured interatomic distance between C8 and heme iron was 2.7 Å (Figure 4A), which allowed the contact of the ferric peroxy anion and led to the electrons’ transfer from the substrate and the subsequent formation of AFBO. Therefore, the conformations in this cluster predicted formation of active metabolite. For the other cluster, on the contrary, the docking result showed that the oxygen atom at C1 of AFB1 was oriented towards the heme iron, whereas C8, C9-double bond was far away from the oxidized center, in which case no AFBO could be formed, thus these conformations were thought inactive. It also can be seen from the putative binding model that AFB1 interacted with residues in the active site by forming two hydrogen bonds: one was between side chain hydroxyl group of Tyr309 and O6 or O7 of AFB1; the other was between the oxygen atom at C4 of AFB1 and the hydrogen in a peptide bond formed by Lys212 with Phe213 (Figure 4A). It is speculated that these two hydrogen bonds ought to play special roles in maintaining the stable binding of AFB1 in the active center.

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.