Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7 |
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Authors: | Valentina Bosello-Travain Marcus Conrad Giorgio Cozza Alessandro Negro Silvia Quartesan Monica Rossetto Antonella Roveri Stefano Toppo Fulvio Ursini Mattia Zaccarin Matilde Maiorino |
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Institution: | 1. Department of Molecular Medicine, University of Padova, Viale G. Colombo, 3, I-35121-Padua, Italy;2. Institute of Developmental Genetics, Deutsches Zentrum für Neurodegenerative Erkrankungen and Helmholtz Zentrum München, Ingolstädter Landstrasse 1, D-85764 Munich-Neuherberg, Germany;3. Department of Biomedical Sciences, University of Padova, Viale G. Colombo, 3, I-35121-Padua, Italy |
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Abstract: | BackgroundMammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.MethodsReducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.ResultsOxidation of the CP is fast (k+ 1 > 103 M− 1 s− 1), however the rate of reduction by GSH is slow (k′+ 2 = 12.6 M− 1 s− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+ 1 > 103 M− 1 s− 1), but not by Trx. By surface plasmon resonance analysis, a KD = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.ConclusionsGPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.General significanceIn the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH. |
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Keywords: | CysGPxs subfamily of glutathione peroxidases containing a Cys redox center 1CysGPx a glutathione peroxidase containing the CP only 2CysGPxs glutathione peroxidases containing both the CP and the CR CP peroxidatic Cys CR resolving Cys GPx7 glutathione peroxidase 7 ER endoplasmic reticulum GSH reduced glutathione GSSG oxidized glutathione GPxs glutathione peroxidases PDI protein disulfide isomerase PCOOH phosphatidylcholine hydroperoxide Prdxs peroxiredoxins ROOH hydroperoxide Sec Selenocysteine SecGPxs subfamily of glutathione peroxidases containing a Sec redox center Syn synuclein Trx thioredoxin TrxR thioredoxin reductase UP peroxidatic Selenocysteine |
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