Molecular Mechanism Study On The Hydroxylation Metabolism Of T-2Toxin By PIG CYP3A29

In trichothecenes, T-2toxin has the strongest toxicity. Eating the feed which is contaminated by the trichothecenes can impair the livestock and make the production decline. This case also poses a potential threat to human health indirectly. The metabolism of T-2toxin is different ranging from different animals to different species. The pig is one of the most important food animals in our country and the most sensitive animal according to the reference. T-2toxin is widely distributed in the corn, oats and mixed feed. It hazards human beings and animals mainly through contaminated food and feed. Exposed to T-2toxin for long will make animals poor feeding and vomiting, leading to collapse, ataxia, shock and even death when the situation is serious. T-2toxin also can inhibit the synthesis of proteins and nucleic acids, interfere the energy metabolism and impair the tissue and cells proliferating rapidly such as skin, intestinal mucosa, white blood cells and so on. Obviously, T-2toxin has a very vital significance in preventing potential hazards to human beings and animals and improving the benefits of animal production.Cytochrome P450enzyme is a class of B family of cytochrome superfamily protease which relies on the heme as cofactor. Cytochrome P450enzyme is one of the most important drug-metabolizing enzymes. It involves the phase I metabolism of exogenous compounds and makes the xenobiotics develop in two directions through the metabolism. One is metabolic detoxification, another is metabolic activation. CYP3A29is one of the most important P450enzymes and has the highest expression in pigs. It also plays a vital role in the metabolism of pigs. The research of P450enzyme is an important aspect of lots of researchs such as veterinary mechanism of cytotoxicity, efficacy occurred, drug discovery and so on. The species and individual differences of drug metabolism can be recognized through this study. What is more, it can suggest basis to consider for veterinary clinical medicine and veterinary drug residue.This study uses bioinformatic techniques to determine the critical amino acid residues that affect CYP3A29metabolizing T-2toxin, then utilize the expression vector pFastBac HTb-CYP3A29constructed by our laboratory for template, and construct mutants by rapid PCR-directed mutagenesis techniques, then use BAC-to-BAC baculovirus expression systems to express the mutants. The expression CYP3A29incubates with nifedipine to measure the activity of CYP3A29, and observe the impact of these mutants metabolizing nifedipine. Finally use electrospray ionization hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography （LC/MS-IT-TOF） to do semi-quantitative research of CYP3A29metabolizing T-2toxin, initially identified the amino acid residues affecting the metabolism between CYP3A29of T-2toxin, then use isothermal titration calorimeter to determine the amino acid residues. Integrate the results, and get the important amino acid sites that affect the function of CYP3A29.1The key anmino acids of CYP3A29in the metabolism of T-2toxinUse human CYP3A4as a template, the homologous modeling software MODELLER utilize CYP3A29modeling to build very similar the resulting structure grossly with CYP3A4. Assess CYP3A29structural model using software Procheck, Verify₃D. The results showed91.5%of the amino acids of this model in the optimum zone,6.9%is in a more suitable area,0.5%is in the acceptable zone,1.1%is irrational zone. The value of Verify₃d is92.46%, which means good compatibility of the tertiary structure of the model and primary structure. Therefore, this model is reasonably available.R106, F108, S119, K212, F213, F215, R372and E374nine amino acid residues found in the interaction of T-2toxin and CYP3A29model using molecular docking software Autodock docking. The R105and K212form hydrogen bonds with the T-2, another amino acid residue were formed the weak interaction of the non-covalent bond with the T-2. Docking simulation results show that, of these amino acid residues on in CYP3A29the active center, the binding T-2and orientation is very important.2The expression of CYP3A29and mutantThe laboratory original pFastBac HTb-CYP3A29as a template, site-directed mutagenesis used the rapid PCR technology to construct mutants, transposited into bacmid then transfected into a baculovirus recombinant virus containing the coding sequence of CYP3A29. CYP3A29protein expressed in Sf9insect cells after baculovirus infected. Western blot validated expression results. The results show successful expression, the specific substrates nifedipine was used to incubated with the expressed protein, followed by HPLC, showing good activity of the expression of CYP3A29and mutants.3The influence of point mutations that CYP3A29metabolism of T-2toxinUse mutants incubation with nifedipine to analyze mutation impact on the CYP3A29metabolism of nifedipine. Then mutants incubated with T-2using liquid chromatography and isothermal titration calorimeter of metabolic situation analysis, assessment mutation on the the CYP3A29metabolic impact of T-2.Kinetic analysis shows that the CYP3A29mutants metabolize nifedipine, R106A can increase CLint value. S119A and K212A can reduce the CLint value, indicating that these amino acid residues closely CYP3A29metabolic capability. Use LC/MS to analyze the metabolism of CYP3A29and T-2, the results show that the mutant R105A, S119A and K212A generate the amount of product is significantly less than the wild-type of CYP3A29. The R106A increase in use of isothermal titration calorimetry analysis found, mutant R106A metabolic T-2heat release a certain increase, and mutants R105A, S119A, and K212A metabolic T-2of the heat release decreased significantly, and the analysis that R105A, S119A, and K212A play a very critical role on CYP3A29metabolic T-2. The combined of CYP3A29metabolic Nifedipine experiment can be speculated that the R105may be the recognition site of the T-2and CYP3A29. S119A and K212A have an important influence on the function CYP3A29.In conclusion, this study using homology modeling and molecular docking methods to identified nine amino acid about CYP3A29metabolism of T-2, and using rapid PCR-directed mutagenesis and baculovirus-insect cell expression system in vitro recombinant expression CYP3A29, and through the liquid phase liquid chromatography and isothermal titration calorimeter mutant metabolism of T-2and nifedipine were analyzed, to some extent, to reveal the relationship between structure and function of the the CYP3A29metabolism of T-2toxin, and molecular catalysis mechanism of CYP3A29. It’s helpful to understand the structure and function of the P450enzymes.