STRUCTURE-FUNCTION STUDY ON PLANT S-NITROSOGLUTATHIONE REDUCTASE FROM TOMATO

David Kopečný^1,2, Lucie Kubienová², Martina Tylichová¹, Pierre Briozzo³, Jana Skopalová⁴, Marek Šebela^1,2, Milan Navrátil⁵, Roselyne Tâche³, Lenka Luhová², Marek Petřivalský²

¹Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research and ²Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic; ³Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Route de St-Cyr, F-78026 Versailles Cedex, France; ⁴Department of Analytical Chemistry, Faculty of Science, Palacký University, 17. Listopadu 12, CZ-771 46, Olomouc, Czech Republic; ⁵Department of Cell Biology and Genetics, Faculty of Science, Palacký University in Olomouc, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic

S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase (EC 1.1.1.284), belongs to the large alcohol dehydrogenase (ADH) superfamily, namely to the ADH3 family. GSNOR catalyzes the oxidation of HMGSH to S-formylglutathione using NAD⁺ as a coenzyme. However, the enzyme catalyzes also the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In general, GSNO serves as a nitrogen oxide (NO) donor in distant tissues and in humans the alternations in the metabolism of GSNO are linked to several lung diseases including asthma. 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 defense. The enzyme thus participates in controlling the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has been recently characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR from tomato (Solanum lycopersicum). The crystal structures of the apoenzyme and the enzyme in complexes with NAD⁺ and NADH with GSH were solved up to 1.9 Å resolution. They represent the first structures within the plant ADH3 family. The crystals were primitive orthorhombic, space group P2₁2₁2 and P2₁2₁2₁, and contained one dimer per asymmetric unit. The calculated Matthews coefficient was ~ 2.4 ų Da^-1, corresponding to a solvent content of 49 %. Each monomer comprises two zinc atoms and consists of a catalytic and a coenzyme domain. The solved structures confirm that coenzyme binding is associated with a zinc movement and changes in its coordination. The first zinc atom functions as a Lewis acid and activates alcohols or other substrates during catalysis while the second one has a structural role. The enzyme preferentially oxidizes HMGSH although some other alcohols such as cinnamylalcohol, geraniol and 12-hydroxydodecanoic acid are also good substrates. In the reductase mode with NADH the enzyme exhibits a high affinity and catalytic efficiency for GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors.

This work was supported by grants from Ministry of Education, Youth and Sports of Czech Republic (LH11013), Czech Science Foundation (GAČR P501/11/1591 and by grant PrF_2011_031 from the Faculty of Science, Palacký University in Olomouc.