The Key Amino-acid Residues Of Porcine CYP3A22 And CYP3A46 Involved In The 3'-Hydroxylation Of T-2 Toxin

Cytochrome P450s(CYPs) belong to the superfamily of heme-thiolate proteins, which consist of a group of structure-function related isoenzymes. They have been identified almost in all biological organisms, and are involved in the oxidation, peroxidation and reduction of endogenous chemicals and the metabolism of exogenous compounds including 90% of clinical medicine and chemicals in the environment.T-2 toxin is one of the most stable and toxic trichothecene mycotoxins, which is found in grains such as maize, wheat and also in food additives. Food intake of feeds contaminated with T-2 toxin may be toxic even lethal to animals. The metabolites of T-2 toxin formed in livestock and poultry may endanger the health of human beings indirectly. Pigs, as one of the livestock cultured and consumed greatly in China, are more vulnerable to T-2 toxin.Hydroxylation is one of the most occurring reactions of T-2 toxin in vivo, which is importantto its toxicity. Previous studies have found that CYP3A subfamily(CYP3A22, CYP3A46 and CYP3A29) might involved in the hydroxylation of T-2 or HT-2 toxin. The key amino acid residues of CYP3A29 but not CYP3A22 and CYP3A46 that involved in the 3'-hydroxylation of T-2 toxin have been preliminarily elucidated.Purpose and Methods: The recombinant proteins were expressed and purified by combination with methods of homologous modeling, molecule docking, site-directed mutagenesis and activity detection assay. We try to get recombinant porcine CYP3A proteins of natural conformation and stable activity, and then screen the key sites, especially in CYP3A22 and CYP3A46 that catalyze hydroxylationof T-2 toxin.Results: 1.By different heterologous expression system optimization, stable and active CYP3A subfamily proteins are finally expressed and obtained in eukaryotic cell expression system, which can hydroxylate T-2 toxin in vitro;2. Key sites of CYP3A22 involved in hydroxylation of T-2 toxin are screened by molecular docking, which are listed below: Met-57,Arg-105,Arg-106, Ser-119,Arg-212, Phe-213,Asp-214, Phe-215,Ala-305, ILe-301, Thr-309, ILe-369,Ala-370,Ala-371,Arg-372, GLy-481 and Ile-483.Among them, Phe-213, Thr-309 and Gly-481 can form hydrogen bonds with T-2 toxin many times. For CYP3A46, key sites are Phe-108, Phe-304, Pro-371,Ala-305, Thr-370, Lys-212, Phe-215, Phe-213,Arg-372,Arg-106,Arg-105, Glu-374 and Leu-373, and Lys-215, Phe-213 and Glu-374 can form hydrogen bonds with T-2 toxin many times.3.Further study on the key sites of CYP3A46 are found that, F213A, F215A and E374A mutants cannot hydroxylate T-2 toxin. Hydroxylation capacity of R106A, F108A and T370A mutants show significant decline compared to wild-type, K212A mutant shows no obvious change, about 75% of wild-type. Taken together,Arg-106, Phe-108, Phe-213, Phe-215, Thr-370 and Glu-374 are the key sites that catalyzehydroxylation of T-2 toxin; Lys-212 may influence T-2 toxin enter into the activity region.