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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.


The circular dichroism spectra and CYP3A29 and the mutants’ nifedipine oxidation activities. (A) Far-UV CD spectra of CYP3A29 and its mutants. CD spectra were recorded at 20 °C and in 50 mM potassium phosphate buffer (200 mM KCl, 0.2 mM DTT, 1 mM EDTA, and 20% glycerol, pH 7.4). (B–G) The HPLC chromatogram of oxidized nifedipine standard; the HPLC chromatogram of nifedipine standard; HPLC chromatograms of nifedipine metabolized by WT, S119A, F304A, and T309A, respectively. HPLC eluent was monitored by absorbance at 254 nm.
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toxins-08-00267-f002: The circular dichroism spectra and CYP3A29 and the mutants’ nifedipine oxidation activities. (A) Far-UV CD spectra of CYP3A29 and its mutants. CD spectra were recorded at 20 °C and in 50 mM potassium phosphate buffer (200 mM KCl, 0.2 mM DTT, 1 mM EDTA, and 20% glycerol, pH 7.4). (B–G) The HPLC chromatogram of oxidized nifedipine standard; the HPLC chromatogram of nifedipine standard; HPLC chromatograms of nifedipine metabolized by WT, S119A, F304A, and T309A, respectively. HPLC eluent was monitored by absorbance at 254 nm.

Mentions: To obtain information about the secondary structure of CYP3A29 and its mutants, we carried out circular dichroism experiments. The far-UV CD spectra of all the proteins displayed two negative peaks at 208 nm and 222 nm (Figure 2A), suggesting typical structures rich in α-helixes. The analysis by CONTINLL software (Colorado State University, Fort Collins, CO, USA, 2000) showed that the recombinant CYP3A29 contained 48.7% α-helix and 9% β-sheet (Table 1), which is similar to the contents of the secondary structure of human CYP3A4 (43% α-helix and 7% β-sheet, data from RCSB Protein Data Bank, 1W0E). The secondary structure contents (45.7% α-helix and 11% β-sheet, Table 1) of S119A were approximately the same as those of WT. Interestingly, compared with the other proteins, F304A contained the minimum α-helix (40.7%) and maximum β-sheet (13.9%), which was exactly the opposite to the case of T309A (52.8% α-helix, 7.9% β-sheet, Table 1).


Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B 1
The circular dichroism spectra and CYP3A29 and the mutants’ nifedipine oxidation activities. (A) Far-UV CD spectra of CYP3A29 and its mutants. CD spectra were recorded at 20 °C and in 50 mM potassium phosphate buffer (200 mM KCl, 0.2 mM DTT, 1 mM EDTA, and 20% glycerol, pH 7.4). (B–G) The HPLC chromatogram of oxidized nifedipine standard; the HPLC chromatogram of nifedipine standard; HPLC chromatograms of nifedipine metabolized by WT, S119A, F304A, and T309A, respectively. HPLC eluent was monitored by absorbance at 254 nm.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-08-00267-f002: The circular dichroism spectra and CYP3A29 and the mutants’ nifedipine oxidation activities. (A) Far-UV CD spectra of CYP3A29 and its mutants. CD spectra were recorded at 20 °C and in 50 mM potassium phosphate buffer (200 mM KCl, 0.2 mM DTT, 1 mM EDTA, and 20% glycerol, pH 7.4). (B–G) The HPLC chromatogram of oxidized nifedipine standard; the HPLC chromatogram of nifedipine standard; HPLC chromatograms of nifedipine metabolized by WT, S119A, F304A, and T309A, respectively. HPLC eluent was monitored by absorbance at 254 nm.
Mentions: To obtain information about the secondary structure of CYP3A29 and its mutants, we carried out circular dichroism experiments. The far-UV CD spectra of all the proteins displayed two negative peaks at 208 nm and 222 nm (Figure 2A), suggesting typical structures rich in α-helixes. The analysis by CONTINLL software (Colorado State University, Fort Collins, CO, USA, 2000) showed that the recombinant CYP3A29 contained 48.7% α-helix and 9% β-sheet (Table 1), which is similar to the contents of the secondary structure of human CYP3A4 (43% α-helix and 7% β-sheet, data from RCSB Protein Data Bank, 1W0E). The secondary structure contents (45.7% α-helix and 11% β-sheet, Table 1) of S119A were approximately the same as those of WT. Interestingly, compared with the other proteins, F304A contained the minimum α-helix (40.7%) and maximum β-sheet (13.9%), which was exactly the opposite to the case of T309A (52.8% α-helix, 7.9% β-sheet, Table 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.