Mechanism of porcine liver xanthine oxidoreductase mediated N-oxide reduction of cyadox as revealed by docking and mutagenesis studies.
Mechanism of porcine liver xanthine oxidoreductase mediated N-oxide reduction of cyadox as revealed by docking and mutagenesis studies.
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对接和诱变研究揭示猪肝黄嘌呤氧化还原酶介导 Cyadox N-氧化物还原的机制
DOI：
10.1371/journal.pone.0073912
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Yuan Z
中科院分区：
文献类型：
--
作者：
Chen C;Cheng G;Hao H;Dai M;Wang X;Huang L;Liu Z;Yuan Z
黄嘌呤氧化还原酶（Xanthine oxidoreductase，XOR）是一种细胞质内含嘌呤的氧化还原酶，催化内源性嘌呤和外源性化合物。这表明，异或在猪肝细胞中催化喹喔啉1，4-二-N-氧化物（QdNOs）的N-氧化物还原。为了阐明这种代谢的分子机制，克隆了猪XOR的cDNA并在草地贪夜蛾昆虫细胞中异源表达。牛的XOR，显示91%的序列同一性，猪的XOR，被用作模板的同源性建模。将QdNOs的代表性化合物cysteine对接到猪异或模型中，定位了8个氨基酸残基，即Gly 47、Asn 352、Ser 360、Arg 427、Asp 430、Asp 431、Ser 1227和Lys 1230，它们与cysteine的距离小于4 π。通过定点突变分析其催化功能。与野生型猪XOR相比，G47 A、S360 P、D431 A、S1227 A和K1230 A在胞嘧啶还原过程中的动力学参数发生了改变，这与黄嘌呤氧化过程中的动力学参数相似，表明这些突变影响了黄嘌呤的供电子过程，然后将电子转移到胞嘧啶以完成N-氧化物还原。而位于424-434环的R427 E和D430 H在还原Cyt时的Km和Vmax分别降低。Arg 427可能与猪XOR与CyR的底物结合有关，Asp 430可能参与电子向CyR的转移。本研究初步揭示了猪XOR在胞嘧啶代谢中的可能催化机制，为XOR在外源性二氮氧化物还原中的结构-功能关系提供了新的见解。
Xanthine oxidoreductase (XOR) is a cytoplasmic molybdenum-containing oxidoreductase, catalyzing both endogenous purines and exogenous compounds. It is suggested that XOR in porcine hepatocytes catalyzes the N-oxide reduction of quinoxaline 1,4-di-N-oxides (QdNOs). To elucidate the molecular mechanism underlying this metabolism, the cDNA of porcine XOR was cloned and heterologously expressed in Spodoptera frugiperda insect cells. The bovine XOR, showing sequence identity of 91% to porcine XOR, was employed as template for homology modeling. By docking cyadox, a representative compound of QdNOs, into porcine XOR model, eight amino acid residues, Gly47, Asn352, Ser360, Arg427, Asp430, Asp431, Ser1227 and Lys1230, were located at distances of less than 4Å to cyadox. Site-directed mutagenesis was performed to analyze their catalytic functions. Compared with wild type porcine XOR, G47A, S360P, D431A, S1227A, and K1230A displayed altered kinetic parameters in cyadox reduction, similarly to that in xanthine oxidation, indicating these mutations influenced electron-donating process of xanthine before subsequent electron transfer to cyadox to fulfill the N-oxide reduction. Differently, R427E and D430H, both located in the 424–434 loop, exhibited a much lower Km and a decreased Vmax respectively in cyadox reduction. Arg427 may be related to the substrate binding of porcine XOR to cyadox, and Asp430 is suggested to be involved in the transfer of electron to cyadox. This study initially reveals the possible catalytic mechanism of porcine XOR in cyadox metabolism, providing with novel insights into the structure-function relationship of XOR in the reduction of exogenous di-N-oxides.
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