Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii
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...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-01-01
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| Series: | Redox Biology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2213231720310119 |
| _version_ | 1819276065774764032 |
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| author | Andrea Tagliani Jacopo Rossi Christophe H. Marchand Marcello De Mia Daniele Tedesco Libero Gurrieri Maria Meloni Giuseppe Falini Paolo Trost Stéphane D. Lemaire Simona Fermani Mirko Zaffagnini |
| author_facet | Andrea Tagliani Jacopo Rossi Christophe H. Marchand Marcello De Mia Daniele Tedesco Libero Gurrieri Maria Meloni Giuseppe Falini Paolo Trost Stéphane D. Lemaire Simona Fermani Mirko Zaffagnini |
| author_sort | Andrea Tagliani |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-23T23:34:18Z |
| format | Article |
| id | doaj.art-4eb3d85e073841f7abbe93374b480235 |
| institution | Directory Open Access Journal |
| issn | 2213-2317 |
| language | English |
| last_indexed | 2024-12-23T23:34:18Z |
| publishDate | 2021-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Redox Biology |
| spelling | doaj.art-4eb3d85e073841f7abbe93374b4802352022-12-21T17:25:54ZengElsevierRedox Biology2213-23172021-01-0138101806Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtiiAndrea Tagliani0Jacopo Rossi1Christophe H. Marchand2Marcello De Mia3Daniele Tedesco4Libero Gurrieri5Maria Meloni6Giuseppe Falini7Paolo Trost8Stéphane D. Lemaire9Simona Fermani10Mirko Zaffagnini11Department of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, Italy; CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, F-75005, Paris, FranceDepartment of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, ItalyCNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, F-75005, Paris, France; CNRS, Institut de Biologie Physico-Chimique, Plateforme de Protéomique, FR550, F-75005, Paris, FranceCNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, F-75005, Paris, FranceDepartment of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, ItalyDepartment of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, ItalyDepartment of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, ItalyDepartment of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, ItalyDepartment of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, ItalyCNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, F-75005, Paris, France; Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, Laboratory of Computational and Quantitative Biology, UMR7238, F-75005, Paris, France; Corresponding author. CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, F-75005, Paris, France.Department of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, Italy; CIRI Health Sciences & Technologies (HST), University of Bologna, I-40064, Bologna, Italy; Corresponding author. Department of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, Italy.Department of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, Italy; Corresponding author.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.http://www.sciencedirect.com/science/article/pii/S2213231720310119ChlamydomonasCysteineNitrosoglutathioneNitrosoglutathione reductaseRedox regulationThiol modification |
| spellingShingle | Andrea Tagliani Jacopo Rossi Christophe H. Marchand Marcello De Mia Daniele Tedesco Libero Gurrieri Maria Meloni Giuseppe Falini Paolo Trost Stéphane D. Lemaire Simona Fermani Mirko Zaffagnini Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii Redox Biology Chlamydomonas Cysteine Nitrosoglutathione Nitrosoglutathione reductase Redox regulation Thiol modification |
| title | Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii |
| title_full | Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii |
| title_fullStr | Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii |
| title_full_unstemmed | Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii |
| title_short | Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii |
| title_sort | structural and functional insights into nitrosoglutathione reductase from chlamydomonas reinhardtii |
| topic | Chlamydomonas Cysteine Nitrosoglutathione Nitrosoglutathione reductase Redox regulation Thiol modification |
| url | http://www.sciencedirect.com/science/article/pii/S2213231720310119 |
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