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Berto, Paola (2009) Espressione omologa ed eterologa della [FeFe ]-idrogenasi di Chlamydomonas reinhardtii. [Tesi di dottorato]

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Abstract (inglese)

Hydrogenases, key enzymes in hydrogen metabolism in several microorganisms, are considered as a possible future energy source. In particular, the ability of the green alga C. reinhardtii to reduce protons and release hydrogen gas (H2) upon illumination by means of a [FeFe]-hydrogenase represents a phenomenon of great scientific interest, because it holds the promise of generating energy from nature’s most plentiful resources: solar energy and water. However, the catalytic activity is strongly and irreversibly inhibited by the molecular oxygen produced during photosynthesis; furthermore, the algal hydrogenase is expressed at very low levels and only in conditions of strict anaerobiosis. This mutually exclusive nature of O2 and H2 photoproduction cannot be easily overcome and represents an important problem in the development of H2 production biotechnology. The study of the structure-function relationship of [FeFe] hydrogenases, which would help to clarify the molecular mechanisms underlying both H2 production and O2 sensitivity, requires the characterization of purified native and modified proteins. The 3D structure of the [FeFe]-hydrogenase from C. reinhardtii has not been solved, mainly because of the very low levels of protein which can be directly purified from the algae. I overexpressed this enzyme in homologous and heterologous systems to obtain enough protein for biochemical studies. The cyanobacterium Synechococcus PCC 7942, which holds a bidirectional [NiFe]-hydrogenase, is able to produce the [FeFe]-hydrogenase from C. pasteurianum in a catalytically active form, thus suggesting that the [NiFe]-hydrogenase maturation pathway of cyanobacteria may be flexible enough to allow the biosynthesis of functional Fe-only enzyme. I obtained two constructs to stably transform the cyanobacterium Synechocystis sp. PCC 6803 and to enable it to express the C. reinhardtii [FeFe]-hydrogenase. The recombinant strains expressing the algal [FeFe]-hydrogenase were able to release H2 gas amounts 4 to 5 times higher than that of wild type strain (which has only a [NiFe]-hydrogenase). These data open new perspectives about the indispensable presence of HydE, HydF and HydG auxiliary proteins to obtain a correctly folded [FeFe]-hydrogenase. At the same time, I proceeded with the homologous overexpression of the [FeFe]-hydrogenase in C. reinhardtii. Since serious limits of this system are the low amount of protein expressed and the instability of mutant algal strain, I am defining new strategies to solve this problem. I will operate site-direct mutations in critical residues to understand the catalytic mechanism and to improve the hydrogen productivity of the enzyme.

Abstract (italiano)

Le idrogenasi, enzimi chiave nel metabolismo dell’idrogeno di molti microrganismi, sono considerate una possibile futura fonte di energia. In particolare, la capacità dell’alga verde C. reinhardtii di ridurre protoni e rilasciare idrogeno gassoso dopo illuminazione per mezzo di una [FeFe]-idrogenasi rappresenta un fenomeno di grande interesse scientifico, poiché permetterebbe di ottenere energia dalle due risorse naturali più diffuse: acqua ed energia solare. Tuttavia, l’attività catalitica è fortemente ed irreversibilmente inibita dall’ossigeno prodotto durante la fotosintesi; inoltre, l’idrogenasi algale è espressa a livelli molto bassi e solo in stretta anaerobiosi. Questa natura mutualmente esclusiva della fotoproduzione di idrogeno ed ossigeno rappresenta un importante ostacolo nello sviluppo della produzione biotecnologica di idrogeno. Lo studio della relazione struttura-fuzione della [FeFe]-idrogenasi, che permetterebbe di chiarire i meccanismi molecolari alla base della produzione di H2 e della sesibilità all’O2, richiede la caratterizzazione della proteina nativa e modificata. La struttura 3D della [FeFe]-idrogenasi di C. reinhardtii non è ancora stata ottenuta, principalmente perché le quantità di proteina purificata direttamente dall’alga sono molto basse. Quindi, ho sovraespresso tale enzima in un sistema omologo ed in uno eterologo per ottenere una quantità di proteina sufficiente per condurre studi biochimici. Il cianobatterio Synechococcus PCC 7942, che possiede una [NiFe]-idrogenasi, è in grado di sintetizzare la [FeFe]-idrogenasi di C. pasteurianum in forma attiva, suggerendo che il sistema di maturazione dei cianobatteri sia suffientemente flessibile da permettere la biosintesi di una [FeFe]-idrogenasi funzionale. Nel corso del dottorato ho ottenuto due costrutti per trasformare il cianobatterio Synechocystis sp. PCC 6803 e permettere l’espressione della [FeFe]-idrogenasi algale. I ceppi ricombinanti esprimenti l’enzima di C. reinhardtii sono in grado produrre idrogeno in quantità da 4 a 5 volte superiori rispetto al ceppo wild type (che ha solo una [NiFe]-idrogenasi). Questi dati aprono nuove prospettive circa l’indispensabile presenza delle proteine accessorie HydE, HydF e HydG per ottenere una [FeFe]-idrogenasi correttamente ripiegata. Contemporaneamente, ho condotto l’espressione omologa dell’enzima direttamente in C. reinhardtii. Dal momento che importanti limiti di questo sistema sono dati dal basso quantitativo di proteina espressa e dall’instabilità del ceppo mutante dell’alga, sto attualmente mettendo a punto nuove strategie per risolvere questi problemi. Inoltre, saranno condotte delle mutagenesi sito-specifiche per chiarire i meccanismi catalitici e migliorare la produttività dell’enzima

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Tipo di EPrint:Tesi di dottorato
Relatore:Giacometti, M. Giorgio
Correlatore:Costantini, Paola
Dottorato (corsi e scuole):Ciclo 21 > Scuole per il 21simo ciclo > BIOCHIMICA E BIOTECNOLOGIE > BIOCHIMICA E BIOFISICA
Data di deposito della tesi:30 Gennaio 2009
Anno di Pubblicazione:30 Gennaio 2009
Parole chiave (italiano / inglese):idrogenasi, chlamydomonas, idrogeno, cianobatteri
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/10 Biochimica
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:1769
Depositato il:30 Gen 2009
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