RT Journal Article SR Electronic T1 Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.08.31.275875 DO 10.1101/2020.08.31.275875 A1 Andrea Tagliani A1 Jacopo Rossi A1 Christophe H. Marchand A1 Marcello De Mia A1 Daniele Tedesco A1 Gurrieri Libero A1 Maria Meloni A1 Giuseppe Falini A1 Paolo Trost A1 Stéphane D. Lemaire A1 Simona Fermani A1 Mirko Zaffagnini YR 2020 UL http://biorxiv.org/content/early/2020/09/02/2020.08.31.275875.abstract AB Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. The kinetic properties of CrGSNOR1 were determined and the high-resolution three-dimensional structures of the apo and NAD+-bound forms of the enzyme were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved 3D-folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed.One-sentence summary GSNOR1 from Chlamydomonas reinhardtii displays an unusual variability of the catalytic zinc coordination environment and an unexpected resistance to thiol-based redox modifications