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  • Recent studies on detailed biochemical and structural charac

    2021-09-13

    Recent studies on detailed biochemical and structural characterization of plant GSNOR ibotenic acid sale confirmed their similarities to mammalian homologues [16], [17], [18], [19]. Compared to human GSNOR, plant GSNORs exhibit differences in the composition of the anion-binding pocket, which negatively influence their affinity for the carboxyl group of hydroxyfatty acids [17]. In Arabidopsis, AtGSNOR1 gene is expressed constitutively and GSNOR protein forms about 0.015% soluble proteins [20]. The possibility of redox regulation of AtGSNOR1 activity has been suggested in detailed bioinformatics study focusing on the role of catalytic and zinc-coordinating Cys residues [18]. Structure of AtGSNOR1 is unusually rich in Cys residues accounting for 3.84% of all residues, compared with an average of 1.37% for all proteins in the UniProt database. Nine non-zinc coordinating cysteine residues are highly conserved in the GSNOR structure, which may be post-translationally modified through S-nitrosation, glutathionylation or reversible oxidation. More specifically, Cys10, Cys271, Cys370 were suggested to have a conserved role in the regulation of GSNOR activity [18]. Recent studies bring additional data supporting the function of conserved non-zinc coordinating cysteines in yeast, plant and mammalian GSNOR as redox switches [21], [22]. S-nitrosation evokes fine conformational changes that alter the shape of the substrate binding cavity and the interaction of GSNOR monomers in the active enzyme dimer. Constitutive GSNOR activity might be negatively regulated in vivo by high levels of nitrosating compounds, thus allowing NO signalling to proceed during NO bursts [22]. Recently, AtGSNOR1 activity was reported to be reversibly inhibited by hydrogen peroxide (H2O2) in vitro and by paraquat-induced oxidative stress in vivo[23]. H2O2-treatment increased the amount of oxidative modifications on Zn2+−coordinating Cys47 and Cys177. Moreover, inhibition of GSNOR resulted in enhanced levels of RSNOs followed by accumulation of GSH. In our previous studies, we characterized in detail GSNOR and its role in plant growth and development in important agricultural plants: tomato (Solanum lycopersicum), cauliflower (Brassica oleracea var. botrytis) and lettuce (L. sativa) [17], [19]. In this study, using selected compounds as NO or nitroxyl (NO−) donors, we show that reductase and dehydrogenase activities of all studied plant GSNORs are inhibited by reversible S-nitrosation of target cysteine residues. Furthermore, we demonstrate plant GSNOR inhibition under oxidative conditions and determined potential S-nitrosation modifications of cysteine residues by LC-MS/MS analysis.
    Materials and methods
    Results and discussion
    Acknowledgements This work was supported by Internal Grant of Palacky UniversityIGA_PrF_2017_016. We thank Elizabeth Vierling and Damian Guerra from University of Massachusetts Amherst for long-term cooperation and fruitful discussions.
    Introduction Similarly to other organisms, nitric oxide (NO) is known as an important gaseous signaling molecule involved in multiple developmental processes of plants and in their responses to various stress stimuli. S-nitrosylation of protein cysteine thiols has been established as one of the major routes of NO action on molecular targets. S-nitrosoglutathione reductase (GSNOR), considered as a key enzyme in the regulation of cellular S-nitrosothiols, belongs to the class III alcohol dehydrogenase family (ADH3, E.C. 1.1.1.1). In older literature, this enzyme was denoted as glutathione-dependent formaldehyde dehydrogenase (GD-FALDH, GS-FDH, E.C. 1.2.1.1). However, as it was elucidated that the true substrate of the enzyme was S-(hydroxymethyl) glutathione (HMGSH), an adduct of glutathione with formaldehyde, the enzyme was renamed to S-(hydroxymethyl) glutathione dehydrogenase (E.C. 1.1.1.284). Within the major formaldehyde detoxification pathway, it catalyzes the NAD+-dependent oxidation of HMGSH to S-formylglutathione. However, the irreversible conversion of GSNO in the presence of NADH resulting in formation of glutathione disulphide (GSSG) and ammonia was later discovered as the physiologically more relevant reaction [27], [32], [39].