N-Oxide Reduction of Quinoxaline-1,4-Dioxides Catalyzed by Porcine Aldehyde Oxidase SsAOX1

Abstract

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/K m values of 1.94 6 0.04, 1.27 6 0.15, and 0.43 6 0.09 minute 21 mM 21, respectively. However, SsAOX1 has the lowest substrate affinity for quinocetone, followed by the cyadox and mequindox, with K m values of 4.36 6 0.56, 3.16 6 0.48, and 2.96 6 0.51 mM, 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.

Estimation of residue depletion of cyadox and its marker residue in edible tissues of pigs using physiologically based pharmacokinetic modelling

“…N-oxide reduction is the predominant metabolic pathway of CYA in pigs (Liu et al, 2009). Xanthine oxidoreductase (XOR) and aldehyde oxidase (AO) are the key enzymes to catalyze the N-oxide reduction of quinoxaline 1,4-di-N-oxides (Liu et al, 2009;Zheng et al, 2011;Mu et al, 2014. Hence, the amount and activity of xanthine oxidoreductase and aldehyde oxidase in liver and intestine determine the rate of CYA metabolism.…”

Differences in the In Vivo and In Vitro Metabolism of Imrecoxib in Humans: Formation of the Rate-Limiting Aldehyde Intermediate

“…The origin of M2 should be defined first because the end-stage metabolite may be directly generated from the parent drug (Xie et al, 2013;Mu et al, 2014) or may undergo successive multistep metabolic pathways (Zhou et al, 2015). In this study, the major metabolic pathway of imrecoxib was characterized as M0 being successively oxidized to M1, to M-CHO, and ultimately to M2, because more M2 was observed in the M1 incubations relative to M0 incubations, and trapping experiments with methoxyamine provided further evidence.…”

The critical role of oxidative stress in the toxicity and metabolism of quinoxaline 1,4-di-N-oxides in vitro and in vivo

“…AO, acridine orange; cCBR1, chicken carbonyl reductase 1; porcine carbonyl reductase 1; SsAOX1, porcine aldehyde oxidase. Cited from (Chen et al, 2013;Huang et al, 2015bHuang et al, , 2015cLi et al, 2014aLi et al, , 2014bLiu et al, 2008Liu et al, , 2009Liu et al, , 2010aLiu et al, , 2010bLiu et al, , 2010cLiu et al, , 2011aLiu et al, , 2011bLiu & Sun, 2013;Mu et al, 2014;Tang et al, 2012;Wang et al, 2016b;Yang et al, 2013a;Zheng et al, 2011). marked as less genotoxicity than other kinds Wang et al, , 2011b.…”

“…The porcine aldehyde oxidase SsAOX1 mainly catalyzed the N!O reduction of QdNOs at the N1 position. SsAOX1 could catalyze QCT, MEQ and CYA to 1-deoxy QCT, 1-deoxy MEQ and 1-deoxy CYA, respectively (Mu et al, 2014).…”