Structure And Function Of Pig Liver Xanthine Oxidoreductase In The Metabolism Of Cyadox

Cyadox is an animal special drug as antimicrobial growth promoters belonging to the quinoxalines family. The early members, such as carbadox and olaquindox, have been widely used in food animal in the last century. However, the late toxicological tests proved that they obviously caused teratogenic, carcinogenic and mutagenic toxicity to animals and human health. The current toxicity studies of cyadox showed no photosensitive, teratogenic, reproductive, and cumulative toxicity in pigs and chickens, which made Cyadox a new alternative in quinoxalines medicine. Now, there are few reports on the metabolic enzymes for the metabolism of cyadox. Since the prototype and metabolites of drugs are responsible for their pharmacological and physical effect, we must research the metabolic enzymes responsible for cyadox metabolism immediately. The study of the key enzyme (s) reponsible for cyadox metabolism not only helps in the approval of the cyadox as veterinary medicine, but also in the correct and effective use of cyadox as a food animal drug, such as formulate reasonable withdrawal period to avoid the emergence of harmful residues and protect the health of human.Xanthine oxidoreductase (XOR) is a molybdenum-containing oxidoreductase, existing in a variety of organic lifes and widely distributed in various tissues of the animals. It is the key metabolic enzymes in the process of hypoxanthine and xanthine metabolism of mammals. XOR is one of the most important drug metabolizing enzymes in addition to the P450enzyme, which is mainly distributed in the cytoplasm and shows the redox activity in the liver cytoplasm of various animals. XOR is mainly involved in the reduction reaction of the nitro, N-oxides, sulfuroxides and other groups. Cyadox1-monoxide and cyadox4-monoxide are the two major metabolites in the metabolism of cyadox in vivo. Furthermore, previous studies have proved that the pig cytosolic proteins were involved in the metabolism of cyadox, suggesting that XOR might play an important role in the metablism of cyadox in the food animals.In this study, XOR specific inhibitor were used to confirm that XOR have abilit to metabolize cyadox. and then pig liver XOR gene was cloned and expressed in the baculovirus system. In order to study the mechanism of metabolization of cyadox by XOR, homology modeling and molecular docking techniques were used to determine the key amino acid residues responsible for cyadox metabolism, and the key residues were mutated by site-directed mutagenesis technology, then the relationship between structure and function about the enzyme and key residues were anayzed in the cyadox metabolism.1. Identification of pig XOR in the metabolism of cyadoxDifferential centrifugation was employed for preparation of liver cytoplasm protein, the metabolites of cyadox and xanthine incubated with cytoplasm in vitro were detected by HPLC, the specific inhibitor of XOR was add to the metabolic system.It was shown that pig liver cytosolic proteins could metabolize cyadox, and the motablism was significantly inhibited by specific inhibitor, indicating that cyadox could be reduced by pig XOR.2. Cloning, expression and activity assay of pig liver XORThe total RNA of liver tissue cells was extracted with trizole. PCR primers were designed based on other XOR CDs sequence to clone pig XOR gene with cDNA as template. pFastBac-XOR recombinant plasmid was constructed to express recombinant protein with baculovirus expression system, which was confirmed by westernblot. The enzymatic kinetic parameters of the recombinant XOR were determined using xanthine and cyadox as substrates.The pig XOR coding sequence was uploaded to the NCBI nucleotide library (serial number: JN896312.1). The recombinant XOR was expressed as active form in sf9insect cell. The Km and Vmax values of recombinant XOR to cyadox are2.7times and0.2times as that of xanthine, respectively. The CLint value of xanthine was nearly15times as that of cyadox. This shows that xanthine has stronger substrate specificity and metabolic activity to XOR than that of cyadox.3. The key anmino acids of pig XOR in the metabolism of cyadoxThe secondary structure of pig XOR was analysed using molecular biology web sites, and homology modeling was conducted with the crystal structure of bovine XOR as a template. Molecular docking between pig XOR as a receptor and cyadox as a ligand was performed to find out the key amino acid residues of cyadox metabolism by XOR.The secondary structure of pig liver XOR is very close to that of bovine XOR. The predicted functional domains are highly conservative, including ironysulfur-center domain (A), FAD domain (B), and Mo-pt domain (C). The DOPE Score of the homology modeling is-157326.5625, and the Verify Score is616.831. The Ramachandran Plot results show that 91.5%amino acid distributed in the optimal region,5.7%amino acids in the credible region,2.8%amino acids in the suspicious region. The cyadox docking with XOR highlighted eight aminoacid of G47, N352, S360, R427, D430, D431, S1227, and K1230in the distance of less than4A between the ligand and receptor significantly. R427, D431, S1227and K1230form hydrogen bond with cyadox, while the others form non-convalent bond with cyadox. These amino acid residues are closely related to the cyadox catalytic process and their subsitutaion may significantly change the metabolic capacity of XOR to cyadox.4. The impact of mutated key amino acids of XOR on the metabolism of cyadoxXOR with mutantion of key amino acids were constructed by site-directed mutagenesis and expressed in sf9insect cell. The metabolism kinetic parameters ofthe mutants were determined, and the relationship between protein structure and function were analyzed.In the xanthine metabolism test, the kinetic analysis results show that D430H and D431A increased the Clint values, and the S360P, S1227A and K1230A reduced the CLint value, indicating these amino acid residues were closely related to xanthine metabolism, which significantly change the metabolism of xanthine ability. At the same time, these mutations changed the enzyme on the substrate affinity and Vmax values, since D431A significantly increased the Vmax of xanthine metabolism, and affinity for xanthine of S360P, S1227A and K1230A were notable reduced, indicating these sites are closely related to substrate binding sites. Pharmacokinetics of cyadox metabolism showed that the cyadox metabolism ability of XOR is less than1/10of xanthine metabolism. The R427E and D431A enhanced CLint value, meaning they could enhance cyadox metabolic capacity. On the contrary, the metabolism of cyadox of S360P, D430H, S1227A and K1230A were decline. The R427E substrate affinity significantly enhanced. The Km value of S360P, S1227A and K1230A significantly increased. In conlusion, all this shows that these amino acid residues are critical with cyadox metabolism.In conclusion, we research the key enzymes in the metabolism of cyadox-xanthine oxidoreductase (XOR)-by vitro recombinant expression, homology modeling and ligand docking of pig liver XOR, and8cyadox metabolism related amino acids were identified in this study. The mutantion provide direct information of related functions and structure relationship in the metabolism of cyadox. The protein function is more visibility by the establish of senior structural model of pig XOR, and the mechanism of catalytic basis on the model. The heterogenously expressed of XOR metabolic system in this study provides a stability of metabolic system for new drug and a scientific research strategy for a new drug research.