Abstract
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.
Highlights
Xanthine oxidoreductase (XOR) is a molybdo-flavoenzyme, existing as two forms, xanthine dehydrogenase (XDH; EC 1.17.1. 4) and xanthine oxidase (XO; EC 1.17.3.2) [1]
In porcine XOR, Glu804 and Arg882 in the active sites of Mo-pt center, and loop 424–434 involved in the conversion from XDH to XO are universally conserved when compared with XORs from other mammals (Figure S1)
The present study has demonstrated that the porcine XOR was expressed in Sf9 cells with XDH/XO activity
Summary
Xanthine oxidoreductase (XOR) is a molybdo-flavoenzyme, existing as two forms, xanthine dehydrogenase (XDH; EC 1.17.1. 4) and xanthine oxidase (XO; EC 1.17.3.2) [1]. Xanthine oxidoreductase (XOR) is a molybdo-flavoenzyme, existing as two forms, xanthine dehydrogenase XOR plays an important role in the catabolism of purines in mammalians. This enzyme catalyzes the oxidation of hypoxanthine to xanthine and further catalyzes the oxidation of xanthine to uric acid. XOR is synthesized as its prevalent XDH form, and can be converted to XO form, either reversibly by disulfide formation or irreversibly by proteolytic cleavage [2]. XOR plays critical roles in the metabolism of exogenous compounds as one of the most important drug-metabolizing enzymes in addition to cytochrome P450 enzymes. In the presence of an adequate electron donor, XOR can mediate the reduction of various compounds, such as nitrobezoic acid, 1-nitropyrene, 2-nitrofluorene, 4-nitroquinoline N-oxide, nitrofurazone and nicotinamide Noxide [1]
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