title: Structure-function analysis of porcine cytochrome P450 3A29 in the hydroxylation of T-2 toxin as revealed by docking and mutagenesis studies.
abstract: T-2 toxin, one of the type A trichothecenes, presents a potential hazard to human and animal health. Our previous work demonstrated that porcine cytochrome P450 3A29 (CYP3A29) played an important role in the hydroxylation of T-2 toxin. To identify amino acids involved in this metabolic process, T-2 toxin was docked into a homology model of CYP3A29 based on a crystal structure of CYP3A4 using AutoDock 4.0. Nine residues of CYP3A29, Arg105, Arg106, Phe108, Ser119, Lys212, Phe213, Phe215, Arg372 and Glu374, which were found within 5 Å around T-2 toxin were subjected to site-directed mutagenesis. In the oxidation of nifedipine, the CLint value of R106A was increased by nearly two-folds compared with the wild-type CYP3A29, while the substrate affinities and CLint values of S119A and K212A were significantly reduced. In the hydroxylation of T-2 toxin, the generation of 3'-OH-T-2 by R105A, S119A and K212A was significantly less than that by the wild-type, whereas R106A slightly increased the generation of 3'-OH-T-2. These results were further confirmed by isothermal titration calorimetry analysis, suggesting that these four residues are important in the hydroxylation of T-2 toxin and Arg105 may be a specific recognition site for the toxin. Our study suggests a possible structure-function relationship of CYP3A29 in the hydroxylation of T-2 toxin, providing with new insights into the mechanism of CYP3A enzymes in the biotransformation of T-2 toxin.
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, China.
authors.forename: Guyue
authors.initials: G
authors.lastname: Cheng
authors.affiliation: National Reference Laboratory of Veterinary Drug Residues (HZAU), MOA Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, China.
authors.forename: Changcun
authors.initials: C
authors.lastname: Liu
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education) at the School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Hongmin
authors.initials: H
authors.lastname: Ma
authors.affiliation: National Reference Laboratory of Veterinary Drug Residues (HZAU), MOA Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, China.
authors.forename: Yuanhu
authors.initials: Y
authors.lastname: Pan
authors.affiliation: National Reference Laboratory of Veterinary Drug Residues (HZAU), MOA Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, China.
authors.forename: Lingli
authors.initials: L
authors.lastname: Huang
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, China.
authors.forename: Haihong
authors.initials: H
authors.lastname: Hao
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, China.
authors.forename: Menghong
authors.initials: M
authors.lastname: Dai
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, China; National Reference Laboratory of Veterinary Drug Residues (HZAU), MOA Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, China.
authors.forename: Zonghui
authors.initials: Z
authors.lastname: Yuan
mesh_terms: D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D062105:Molecular Docking Simulation; D016296:Mutagenesis; D013329:Structure-Activity Relationship; D013552:Swine; D013605:T-2 Toxin
chemical_list: D003577:Cytochrome P-450 Enzyme System; D013605:T-2 Toxin