TY - JOUR T1 - <em>N</em>-Oxide Reduction of Quinoxaline-1,4-Dioxides Catalyzed by Porcine Aldehyde Oxidase SsAOX1 JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 511 LP - 519 DO - 10.1124/dmd.113.055418 VL - 42 IS - 4 AU - Peiqiang Mu AU - Ming Zheng AU - Ming Xu AU - Yuanming Zheng AU - Xianqing Tang AU - Yufan Wang AU - Kaixin Wu AU - Qingmei Chen AU - Lijuan Wang AU - Yiqun Deng Y1 - 2014/04/01 UR - http://dmd.aspetjournals.org/content/42/4/511.abstract N2 - Quinoxaline-1,4-dioxides (QdNOs) are a class of quinoxaline derivatives that are widely used in humans or animals as drugs or feed additives. However, the metabolic mechanism, especially the involved enzymes, has not been reported in detail. In this study, the N-oxide reduction enzyme, porcine aldehyde oxidase SsAOX1 was identified and characterized. The SsAOX1 gene was cloned from pig liver through reverse-transcription polymerase chain reaction using degenerate primers, which encode a 147-kDa protein with typical aldehyde oxidase motifs, two [2Fe-2S] centers, a flavin adenine dinucleotide (FAD) binding domain, and a molybdenum cofactor domain. After heterologous expression in a prokaryote, purified SsAOX1 formed a functional homodimer under native conditions. Importantly, the SsAOX1 catalyzed the N-oxide reduction at the N1 position of three representative QdNOs (quinocetone, mequindox, and cyadox), which are commonly used as animal feed additives. SsAOX1 has the highest activity toward quinocetone, followed by mequindox and cyadox, with kcat/Km values of 1.94 ± 0.04, 1.27 ± 0.15, and 0.43 ± 0.09 minute−1 μM−1, respectively. However, SsAOX1 has the lowest substrate affinity for quinocetone, followed by the cyadox and mequindox, with Km values of 4.36 ± 0.56, 3.16 ± 0.48, and 2.96 ± 0.51 μM, respectively. In addition, using site-directed mutagenesis, we found that substitution of glycine 1019 with threonine endows SsAOX1 with N-oxide reductive activity at the N4 position. The goal of this study was to identify and characterize the N-oxide reduction enzyme for a class of veterinary drugs, QdNOs, which will aid in the elucidation of the metabolic pathways of QdNOs and will provide a theoretical basis for their administration and new veterinary drug design. ER -