S-nitrosoglutathione reductase (GSNOR) is considered a key enzyme in the regulation of intracellular levels of S-nitrosoglutathione and protein S-nitrosylation. As a part of nitric oxide catabolism, GSNOR catalyzes the irreversible decomposition of GSNO to oxidized glutathione. GSNOR is involved in the regulation of plant growth and development, mediated by NO-dependent signaling mechanisms, and is known to play important roles in plant responses to various abiotic and biotic stress conditions. Here we present optimized protocols to determine GSNOR enzyme activities in plant samples by spectrophotometric measurements and by activity staining after the native polyacrylamide gel electrophoresis.
S-nitrosylation of protein cysteine thiol groups has recently emerged as a widespread and important reversible post-translational protein modification, involved in redox signalling pathways of nitric oxide and reactive nitrogen species. S-nitrosoglutathione reductase (GSNOR), member of class III alcohol dehydrogenase family (EC 1.1.1.1), is considered the key enzyme in the catabolism of major low molecular S-nitrosothiol, S-nitrosoglutathione, and hence to control the level of protein S-nitrosylation. Changes of GSNOR activity after exposure to different abiotic stress conditions, including low and high temperature, continuous dark and de-etiolation, and mechanical injury, were investigated in important agricultural plants. Significantly higher GSNOR activity was found under normal conditions in leaves of Cucumis spp. genotype sensitive to biotrophic pathogen Golovinomyces cichoracearum. GSNOR activity was generally increased in all studied plants by all types of stress conditions. Strong down-regulation of GSNOR was observed in hypocotyls of etiolated pea plants, which did not recover to values of green plants even 168 h after the transfer of etiolated plants to normal light regime. These results point to important role of GSNOR during normal plant development and in plant responses to several types of abiotic stress conditions.
- MeSH
- aldehydoxidoreduktasy metabolismus MeSH
- Ascomycota patogenita MeSH
- Cucumis melo enzymologie genetika mikrobiologie MeSH
- Cucumis sativus enzymologie genetika mikrobiologie MeSH
- fyziologický stres * MeSH
- hrách setý enzymologie mikrobiologie MeSH
- hypokotyl enzymologie MeSH
- mechanický stres MeSH
- nízká teplota MeSH
- reakce na tepelný šok MeSH
- světlo MeSH
- vývoj rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase, belongs to the large alcohol dehydrogenase superfamily, namely to the class III ADHs. GSNOR catalyses the oxidation of HMGSH to S-formylglutathione using a catalytic zinc and NAD(+) as a coenzyme. The enzyme also catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In plants, GSNO has been suggested to serve as a nitric oxide (NO) reservoir locally or possibly as NO donor in distant cells and tissues. NO and NO-related molecules such as S-nitrosothiols (S-NOs) play a central role in the regulation of normal plant physiological processes and host defence. The enzyme thus participates in the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has recently been characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR, that from tomato (Solanum lycopersicum). SlGSNOR gene expression is higher in roots and stems compared to leaves of young plants. It is highly expressed in the pistil and stamens and in fruits during ripening. The enzyme is a dimer and preferentially catalyses reduction of GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors. Using NAD(+), the enzyme oxidizes HMGSH and other alcohols such as cinnamylalcohol, geraniol and ω-hydroxyfatty acids. The crystal structures of the apoenzyme, of the enzyme in complex with NAD(+) and in complex with NADH, solved up to 1.9 Å resolution, represent the first structures of a plant GSNOR. They confirm that the binding of the coenzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the well characterized human GSNOR, plant GSNORs exhibit a difference in the composition of the anion-binding pocket, which negatively influences the affinity for the carboxyl group of ω-hydroxyfatty acids.
- MeSH
- aldehydoxidoreduktasy chemie genetika metabolismus MeSH
- apoenzymy chemie genetika metabolismus MeSH
- glutathion metabolismus MeSH
- katalytická doména MeSH
- klonování DNA MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- NAD metabolismus MeSH
- oxidace-redukce MeSH
- regulace genové exprese u rostlin MeSH
- sekvence aminokyselin MeSH
- Solanum lycopersicum enzymologie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
S-Nitrosation is a new type of protein posttranslational modification. S-nitrosothiols, which are stable and mobile reservoirs of nitric oxide, are considered convergence points of signalling pathways of reactive oxygen and nitrogen species. This review summarizes the current knowledge of the origin, properties and biological functions of both low-molecular-weight and protein S-nitrosothiols. The biological functions of the former nitrosothiols are derived from their ability to decompose yielding NO or to trans-nitrosate protein thiols. Altered levels of the S-nitrosothiols and S-nitrosation of proteins are associated with nitrosation stress, resulting in a series of pathological processes. Although considerable advances in the research on S-nitrosothiols and protein S-nitrosation have been recently achieved, understanding the exact role and functions of S-nitrosation in signalling pathways of reactive nitrogen species is still very limited.
