Human Carbonyl Reductase 1 Is an S-Nitrosoglutathione Reductase

Abstract

Human carbonyl reductase 1 (hCBR1) is an NADPH-dependent short chain dehydrogenase/reductase with broad substrate specificity and is thought to be responsible for the in vivo reduction of quinones, prostaglandins, and other carbonyl-containing compounds including xenobiotics. In addition, hCBR1 possesses a glutathione binding site that allows for increased affinity toward GSH-conjugated molecules. It has been suggested that the GSH-binding site is near the active site; however, no structures with GSH or GSH conjugates have been reported. We have solved the x-ray crystal structures of hCBR1 and a substrate mimic in complex with GSH and the catalytically inert GSH conjugate hydroxymethylglutathione (HMGSH). The structures reveal the GSH-binding site and provide insight into the affinity determinants for GSH-conjugated substrates. We further demonstrate that the structural isostere of HMGSH, S-nitrosoglutathione, is an ideal hCBR1 substrate (K m ‫؍‬ 30 M, k cat ‫؍‬ 450 min ؊1 ) with kinetic constants comparable with the best known hCBR1 substrates. Furthermore, we demonstrate that hCBR1 dependent GSNO reduction occurs in A549 lung adenocarcinoma cell lysates and suggest that hCBR1 may be involved in regulation of tissue levels of GSNO.Human carbonyl reductase 1 (hCBR1), 3 an NADPH-dependent enzyme belonging to the short chain dehydrogenase/reductase family, has been shown to be involved in the metabolism of structurally diverse carbonyl-containing substances. This is in contrast to other members of the short chain dehydrogenase/ reductase family, such as 11␤-hydroxysteroid dehydrogenases 1 and 2 that interconvert cortisone and cortisol, and the 17␤-hydroxysteroid dehydrogenases, which have well defined androgen and estrogen substrates. Previously reported substrates of hCBR1 include prostaglandins and xenobiotics such as the anti-cancer anthracyclin doxorubicin and the vitamin K2 precursor menadione (1). More recently, hCBR1 has been linked to the detoxification of reactive aldehydes such as 4-oxonon-2-enal and its GSH conjugate that are believed to play a central role in oxidative stress-related neurodegenerative disorders including Alzheimer and Parkinson diseases (2). Although extensive biological investigations of hCBR1, including RNA interference, pharmacology, and crystallography of human hCBR1 in complex with substrate mimics, have been carried out (3), the endogenous physiological substrate(s) of this enzyme remain to be defined. One well accepted aspect of hCBR1 substrate recognition is the presence of a GSH-binding pocket predicted to be in close proximity to the catalytic site. GSH conjugates of otherwise poorly recognized substrates such as prostaglandin A1 are reduced by hCBR1 (4), supporting this hypothesis. To identify additional physiological substrates of hCBR1, we initiated a structural biology effort to analyze the GSH-binding site of hCBR1, hypothesizing that cellular GSH adducts might serve as particularly good hCBR1 substrates.We solved the x-ray co-crystal structure of hCBR1 in complex with G...

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