Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B₁.

Jun Wu,Peiqiang Mu,Qingmei Chen,Jikai Wen,Jun Jiang,Yiqun Deng,Lijuan Wang,Weiying Xu,Ruohong Chen,Caihui Zhang,Kangbai Li

doi:10.3390/toxins8090267

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.

Highlights

Aflatoxins are secondary metabolites mainly produced by Aspergillus flavus and Aspergilus parasiticus [1]
We demonstrated that the recombinant CYP3A29 expressed in E. coli was capable of transforming aflatoxin B1 (AFB1) into AFBO
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

Summary

Introduction

Aflatoxins are secondary metabolites mainly produced by Aspergillus flavus and Aspergilus parasiticus [1]. They are highly toxic and carcinogenic to animals and humans. Aflatoxin B1 (AFB1 ) is the most toxic and widely distributed of all identified aflatoxins [3,4]. AFB1 is not toxic in its native form, but highly activated when converted to electrophilic AFB1 exo-8,9-epoxide (AFBO) in vivo [5,6]. AFBO is highly reactive and intercalates into DNA, resulting in the genotoxicity of AFB1 [7]. Extensive research has confirmed that CYP3A4 and CYP1A2 play central roles in the bioactivation of AFB1 in human [8,9,10], though there remain some controversies about their relative dominance during this process [10,11,12,13]

Results

Discussion

Conclusion