Cysteine-Based Redox Modifications in the Regulation of Calvin-Benson Cycle Enzymes from Chlamydomonas Reinhartdtii

Di Giacinto, Nastasia (2016) Cysteine-Based Redox Modifications in the Regulation of Calvin-Benson Cycle Enzymes from Chlamydomonas Reinhartdtii, [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Biologia cellulare e molecolare, 28 Ciclo. DOI 10.6092/unibo/amsdottorato/7647.
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In photosynthetic organisms the redox-dependent modification of thiols belonging to enzymes of the photosynthetic cycle, plays a prominent role in the regulation of metabolism and signalling. An important regulatory mechanism is represented by light that acts through a ferredoxin-thioredoxin system. The system permits the reduction/oxidation of disulfide bridges of the target enzymes. Moreover, cysteines residues can undergo other modifications such as glutathionylation and nitrosylation. Proteomic studies have allowed the identification of new putative targets of redox modifications, including Calvin Benson cycle enzymes. The aim of the studies was to investigate whether phosphoglycerate kinase, triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii undergo redox modifications. All enzymes and their mutants (i.e. cysteines variants) were purified and the treatments with alkylating and oxidative agents have permitted to confirm the presence of reactive cysteine(s). The sensitivity of recombinant proteins to redox modifications and the cysteine(s) involved were analyzed by biochemical approcheas. The structural features were analyzed, and the crystallography structure of CrTPI and CrGPA were solved. The three enzymes result all redox regulated although with different biochemical features. The CrPGK contains two cysteines sensitive to redox treatments, although the inhibitor effects of these modifications are different, indeed glutathionylation slightly affected the enzymatic activity compare to nitrosylation. Moreover, the crystallographic structure of CrTPI was determined at a resolution of 1.1.Å, showing a homodimeric conformation containing the typical α/β- barrel fold. No evidence for CrTPI Trx-dependent regulation was obtained but was found to undergo glutathionylation and nitrosylation with a moderate down-regulation on activity. Furthermore, the CrGAPA shows an extreme sensitivity to oxidant molecules and the crystallographic structure of CrGAPA was determined at a resolution of 1.8.Å, confirming the tetrameric fold of the enzyme. the results suggest that redox modifications could constitute a mechanism for the regulation of the Calvin-Benson cycle under oxidative stress conditions.

Tipologia del documento
Tesi di dottorato
Di Giacinto, Nastasia
Dottorato di ricerca
Scuola di dottorato
Scienze biologiche, biomediche e biotecnologiche
Settore disciplinare
Settore concorsuale
Parole chiave
cysteine, Calvin-Benson cycle, S-glutathionylation, S-nitrosylation, Chlamydomonas reinhardtii
Data di discussione
22 Aprile 2016

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