Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells

• Published in: The Journal of biological chemistry 2013-06, Vol.288 (26), p.19221-19237
• Main Authors: Yang, Shaojun, Jan, Yi-Hua, Gray, Joshua P., Mishin, Vladimir, Heck, Diane E., Laskin, Debra L., Laskin, Jeffrey D.
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - chemistry; Animals; Biopterins - analogs & derivatives; Biopterins - chemistry; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Enzymology; Epithelial Cells - cytology; Genetic Vectors; Humans; Lung - cytology; Mice; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Oxidation-Reduction; Oxidative Stress; Quinone Redox Cycling; Quinones; Quinones - chemistry; Reactive Oxygen Species; Reactive Oxygen Species (ROS); Recombinant Proteins - chemistry; Reductase; Tetrahydrobiopterin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.402164

In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury. Background: Enzymes mediating chemical redox cycling in the lung are poorly defined. Results: Sepiapterin reductase was identified as a key mediator of redox cycling and was analyzed using inhibitors and site-directed mutagenesis. Conclusion: Sepiapterin reductase generates reactive oxygen species during redox cycling in a mechanism distinct from sepiapterin reduction. Significance: This is the first report demonstrating that sepiapterin reductase mediates chemical redox cycling.