Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii

Abstract

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. Its kinetic properties were determined and the three-dimensional structures of the apo-, NAD + - and NAD + /GSNO-forms were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved 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 and folding, 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.

Quantitative Proteome Profiling of a S-Nitrosoglutathione Reductase (GSNOR) Null Mutant Reveals a New Class of Enzymes Involved in Nitric Oxide Homeostasis in Plants

“…As shown in Figure 5C , we observed the lowest specific activity for AKR4C8, whereas the other AKR4Cs were more efficient, with three- to fourfold higher activities than those measured in the presence of GSNO. Altogether, these results demonstrate that AKR4Cs are capable of degrading GSNO, but with opposite specificity toward the cofactor (NADPH vs. NADH) and with 20- to 60-fold lower specific activities than plant GSNORs ( Guerra et al, 2016 ; Tagliani et al, 2021 ). Moreover, SNO-CoA appears as a suitable substrate for all A. thaliana AKR4Cs as observed for human AKR1A1 ( Stomberski et al, 2019a ).…”

“…S -nitrosoglutathione and CoA-SNO were prepared freshly as described previously ( Stomberski et al, 2019b ; Tagliani et al, 2021 ). Enzyme assays were performed in 100 mM Tris–HCl (pH 7.9) containing 0.2 mM NAD(P)H, 0.4 mM GSNO or SNO-CoA, and variable amounts of AKR4C proteins (50–200 nM) or A. thaliana leaf protein extracts (50–200 μg).…”

Structural and biochemical characterization of Arabidopsis alcohol dehydrogenases reveals distinct functional properties but similar redox sensitivity

“…To further investigate the redox sensitivity of ADH1 and GSNOR, we examined the effect of the thiol‐modifying agent MMTS, which can reversibly react with both accessible cysteines and metal‐coordinating cysteine thiols (Tagliani et al., 2021). As shown in Figure 6b,c, both enzymes are highly sensitive, and exposure to 1 m m MMTS resulted in almost complete inhibition of enzyme activity after incubation for 5 min (ADH1) or 15 min (GSNOR).…”

Structural snapshots of nitrosoglutathione binding and reactivity underlying S-nitrosylation of photosynthetic GAPDH

“…CrGAPA was treated for 30 min with 1 mM GSNO and MALDI-TOF mass spectrometry analysis was performed before and after incubation with 20 mM DTT for 30 min. The samples were analyzed as described in (31,44).…”