Molecular Mechanism Of Porcine Cytochrome P450 3A Subfamily In The Metabolism Of Aflatoxin B₁

Aflatoxin B₁（AFB₁）is the most toxic form of aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus.It can be catalyzed by cytochrome P450（CYP）in vivo to produce the genotoxic metabolite aflatoxin-8,9-epoxide（AFBO）.AFBO can induce cancer by combining with DNA to cause mutations.At the same time,AFBO can also bind to intracellular proteins,leading to cell death.Therefore,AFB₁ is defined as a class I carcinogen.The CYP3A subfamily plays an important role in the metabolism of AFB₁.Livestock and poultry are more at risk,because they have higher probabilities of being exposed to AFB1 through contaminated feedstuff.Pigs as an indispensable part of the animal husbandry,used to study the metabolism of drugs and other exogenous compounds getting more and more attention.It has been reported that human CYP3A family and porcine CYP3A29 play important roles in the activation of AFB₁ in vivo.However it is unclear whether AFB₁ can be metabolized by other porcine CYP3A subfamily members.In order to understand the molecular mechanism of AFB₁ metabolism in swine CYP3A subfamily,we tried to use E.coli prokaryotic expression system to obtain recombinant CYP3A22 and CYP3A46 proteins.CYP3A22 protein was not obtained,although we tried different expression conditions and transformation strategies.We successfully obtained recombinant CYP3A46 protein,by codon optimization and N-terminal renovation.Furthermore,the recombinant CYP3A46 protein has good structure and activity,determine by circular dichroism,CYP common reduction spectrum and in vitro incubation with nifedipine,a model substrate of CYP3A.And through in vitro incubation experiments,we demonstrated that porcine CYP3A46 can metabolize AFB₁ to produce AFBO for the first time.In order to study the molecular mechanism of CYP3A46 metabolizing AFB_1,the three-dimensional model of CYP3A46 was constructed using online server SWISS-MODEL,with the crystal structure of CYP3A4 as model.The ligand AFB₁ was docking into the three-dimensional model of CYP3A46 using the molecular docking software AutoDock 4.2.The possible conformations were screened by the clustering analysis,and the amino acid residues that may play a role in the metabolic process were analyzed by PyMOL.We constructed and expressed F108A,S119A,K212A,F215,F304A and T309A mutant proteins.By experiments,it was found that the metabolic activity of the F108A and F215A mutants was reduced to 1/3 of the WT.The activity of S119A and T309A also decreased about 70%of WT.Unexpectedly,the mutant F304A increased metabolic activity about 2 fold of the WT.By measuring the enzyme kinetic parameters,we found that the S119 site may effect the cooperation in the process of AFB₁ metabolized by CYP3A46.Unlike the F304A mutant of human CYP3A4,which cause a complete loss of catalytic activity,we found that the F304A of CYP3A46 had a 2-fold increase in AFB₁activity.Based on our molecular docking results,we speculated that the F304 of CYP3A46may exist as a sterically hindered.When F304 was mutated to alanine,AFB₁ formed a hydrogen bond with S119 to promote the catalytic reaction.This speculation was also confirmed in F304W and S119A/F304A.The T309 site may form a hydrogen bond with AFB₁ to stabilize the binding of AFB₁.We also examined the activation of AFB₁metabolism withα-naphthoflavone as an effector,and found thatα-naphthoflavone can stimulated the AFB₁ metabolism activity by porcine CYP3A46.By comparing the multiples of activation,we found thatα-naphthoflavone may effectie the I helix of CYP3A46.At the same time,it was found that the AFB₁ metabolism activity of CYP3A29 and CYP3A46 was significantly different.Through molecular docking and sequence comparison,it was found that the differences in the amino acid threonine and alanine at position 370 may play an important role.Based on the results of our molecular docking and protein activity assays,it was speculated that the 370-position amino acids may affect the metabolic activity of AFB₁by affecting the cooperation of the two AFB₁ molecules.