Capasso, Giorgia (2023) Enzymatic and structural characterization of Glucose-6P Dehydrogenase (G6PDH) from Chlamydomonas reinhardtii. [Tesi di dottorato]
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| Tipologia del documento: | Tesi di dottorato |
|---|---|
| Lingua: | Italiano |
| Titolo: | Enzymatic and structural characterization of Glucose-6P Dehydrogenase (G6PDH) from Chlamydomonas reinhardtii |
| Autori: | Autore Email Capasso, Giorgia giorgia.capasso@unina.it |
| Data: | 6 Marzo 2023 |
| Numero di pagine: | 152 |
| Istituzione: | Università degli Studi di Napoli Federico II |
| Dipartimento: | Biologia |
| Dottorato: | Biologia |
| Ciclo di dottorato: | 35 |
| Coordinatore del Corso di dottorato: | nome email Esposito, Sergio sergio.esposito@unina.it |
| Tutor: | nome email Esposito, Sergio [non definito] |
| Data: | 6 Marzo 2023 |
| Numero di pagine: | 152 |
| Parole chiave: | Cysteine, Oxidative pentose phosphate pathway,Thioredoxins, Redox regulation, Abiotic stress, G6PDH, pET315b, NADPH, Inhibition, Green algae, Plastid |
| Settori scientifico-disciplinari del MIUR: | Area 05 - Scienze biologiche > BIO/04 - Fisiologia vegetale |
| Depositato il: | 20 Mar 2023 09:51 |
| Ultima modifica: | 10 Apr 2025 14:14 |
| URI: | http://www.fedoa.unina.it/id/eprint/15194 |
Abstract
Glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) is the main regulatory enzyme of the oxidative pentose phosphate pathway (OPPP), an important provider of NADPH and these reductants are utilized in both nitrogen assimilation and stress response. Purified plant and algal G6PDHs showed a great instability, thus making difficult the description of their biochemical and physio-structural properties. Thus, an extensive biochemical (and structural) description of the algal plastidial G6PDHs is desirable: therefore, in this project the recombinant plastidial G6PDH from the green alga Chlamydomonas reinhardtii was overexpressed in bacteria, purified, and biochemically characterised. Purified Cr-G6PDH reacted against anti potato P1-G6PDH antibody and showed peculiar kinetic properties. CrG6PDH catalytic activity was inactivated in vitro by reduction. In vivo, this inhibition could be exerted by Trx m: the data here shown demonstrate a sudden and strong inhibition of the activity by Trx m, over 85% after 1 min. The measurement of the enzyme redox potential supported the notion that CrTrx m is the main regulator of CrG6PDH. Purified CrG6PDH showed hyperbolic kinetics versus G6P and NADP+ (KmG6P= 0.315 mM; KmNADP+= 115 µM) and a mixed inhibition by NADPH (KiNADPH= 138 µM). Furthermore, CrG6PDH reaction is strictly dependent by Mg++ cations (KmMg++= 25 μM). This parameter could be potentially interesting because of Mg++ in the stabilization of CrG6PDH structure, possibly facilitating NADP+ binding. Furthermore, a kinetic analysis suggests that the enzyme obeys to a sequential reaction mechanism. Furthermore, post-translational modifications (PTMs) on cysteines were investigated. Thiol-modifying treatments with diamide (TMAD) and DTNB suggested that CrP1-G6PDH contains cysteine(s) that might be prone to oxidative modifications that may affect enzyme catalysis. The activity of CrG6PDH was modulated by PTMs, including S-nytrosilation, H2O2-dependent oxidative treatment and alkylation. Further study will aim to obtain the 3D structure of the CrP1-G6PDH to clarify the allosteric features of G6PDH and their peculiarity in algal G6PDH regulation.
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