Molecular Mechanism Of Porcine Cytochrome P450 2A19 In The Metabolism Of Aflatoxin B₁

Aflatoxin B1(AFB1)is a secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus in the process of contaminating corn,peanut,wheat,barley and other crops,feed and food.It has no clear physiological function,and its metabolism reaction in liver to generate AFB1-8,9-epoxide(AFBO)is the key way to activate its toxicity.Pigs are sensitive to AFB1 and far higher than that of humans and other animals to exposure to AFB1,so the poisoning of aflatoxin is a great harm to pig breeding industry.However,for a long time,compared with viral diseases,bacterial diseases,parasitic diseases and other epidemic infectious diseases,researchers have neglected aflatoxin poisoning in pigs,which is a common non-Infectious disease in livestock and poultry,leading to unclear understanding of its metabolic activation process and lack of prevention and control methods.Therefore,the identification of the key enzyme responsible for catalyzing the metabolism of AFB1 into AFBO in pig liver can not only clarify the pathogenic and toxicological mechanisms of AFB1,but also reduce the formation of toxic metabolites of AFB1 by regulating the enzyme activity,so the study is of great significance to fundamentally reduce the toxicity and harm of AFB1.Cytochrome P450(CYP)is a superfamily of heme-thiolate proteins and is the most critical enzyme that catalyzes the phase I metabolism of AFB1 and affects its toxicity.At present,the identified CYP subtypes that catalyze the metabolism of AFB1 are mainly 1A,2A,and 3A families,which can metabolize AFB1 to produce products such as AFBO,AFM1,and AFQ1.In this study,the porcine CYP2A19 protein was obtained through a prokaryotic expression system,and Fe2+·CO reduction spectra were used to confirm its good activity.After vitro metabolic tests were performed,the results show that porcine CYP2A19 can metabolize coumarin,a model substrate of CYP2A,to generate 7-hydroxycoumarin,and it is the first time to prove that porcine CYP2A19 can metabolize AFB1,mainly producing AFBO and a small amount of AFM1/AFQ1.Compared with porcine CYP1A2,CYP3A29 and CYP3A46 that have been identified as capable of metabolizing AFB1,porcine CYP2A19 metabolizes AFB1 more strongly to produce AFBO,suggesting that it may be the CYP subtype in pig liver that is mainly responsible for the metabolism of AFB1 to AFBO.In order to study the molecular mechanism of porcine CYP2A19 metabolism of AFB1,this study used homology modeling and molecular docking to initially determine the key amino acid sites Phe-107,Ala-117,Phe-209,Asn-297 and Thr-305.The prokaryotic expression system was used to obtain mutant proteins and the enzyme kinetic parameters were measured.The results show that Phe-107 may increase the affinity of the AFB1 molecule in the protein substrate cavity and the active center to increase its affinity for enzymes during the metabolic process,and further improve its catalytic metabolism activity.The disappearance of benzene ring in the side chain of Phe-209 does not affect the conformation of porcine CYP2A19 metabolizing AFB1 to produce AFBO,but it is more conducive to the production of less toxic hydroxylated AFM1 or non-toxic AFQ1.Asn-297 and Thr-305 assist catalytic metabolism by forming hydrogen bonds with the substrate,where the hydrogen bonding force formed between Asn-297 and the substrate plays a decisive role in the binding and catalysis of the substrate in the substrate cavity of enzyme,the hydrogen bond formed between Thr-305 and substrate may only serve to stabilize the ligand.During the metabolism of AFB1 by human CYP2A13 and porcine CYP2A19,the alanine at position 117 plays an important role and its change in the polarity of the amino acid at this position may cause the shape of protein substrate cavity to change,which in turn affects the form of hydrogen bonding in the metabolic process.In summary,this study identified that porcine CYP2A19 metabolizing AFB1 mainly produces AFBO and a small amount of AFM1/AFQ1,and its activity of metabolizing AFB1 to produce AFBO is stronger than porcine CYP1A2,CYP3A29 and CYP3A46,suggesting that it may be mainly responsible for the metabolism of AFB1 in porcine liver CYP subtype of AFBO.Identified key amino acid sites affecting the activity of the enzyme can provide important references and targets for screening porcine strains resistant to AFB1 through genetic breeding,and give new ideas for preventing and controlling swine aflatoxin poisoning.