Biochemical and Structural Characterization of the Chloroplastic Enzyme Transketolase from the Green Microalga Chlamydomonas Reinhardtii

Pasquini, Miriam (2017) Biochemical and Structural Characterization of the Chloroplastic Enzyme Transketolase from the Green Microalga Chlamydomonas Reinhardtii, [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Biologia cellulare e molecolare, 29 Ciclo. DOI 10.6092/unibo/amsdottorato/7987.
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The biochemical and structural properties of the chloroplastic transketolase from Chlamydomonas reinhardtii (CrTK) were investigated, with emphasis on the role of its cofactors, the thiamine pyrophosphate (TPP) and the magnesium, and on its redox regulation involving thiol-based Post-Translational Modifications (PTMs). We overexpressed the recombinant protein and purified it until homogeneity. Its activity depends on a slow reconstitution process in the presence of saturating concentrations of both the cofactors, since the Mg2+ has to ensure the correct orientation/allocation of the TPP in the CrTK active site(s). Interestingly, the activity of the holo-form (CrTKTPP/Mg) is almost unaffected by DTTox (oxidized dithiotreitol) induced oxidation, whereas CrTKapo (apo-form) or CrTKTPP (protein reconstituted in absence of magnesium) show a strong sensitivity to the treatment, due to the induction of a disulfide bond between Cys470 and Cys484. For these reasons, we hypothesize that the role of the Mg2+ and the formation of the disulfide are synergistically addressed to the control of the CrTK redox sensitivity. These features could be related to the physiological increase of the cation concentration in the chloroplasts’stroma and to the thioredoxin-driven activation of the Calvin cycle enzymes during dark-to-light transition, the main events allowing the “awakening” of the Calvin cycle enzymes. Moreover, the influence of the TPP-related molecules on the transketolase (TRK1) gene expression was investigated in Chlamydomonas coltures through qRT-PCR; surprisingly, the presence of the TPP-like compounds led to a decrease in the TRK1 expression levels. The investigation of the redox modifications of CrTK should continue analyzing what type(s) of thioredoxins (TRXs) could act on the CrTK activation, trying to give a further physiological significance to the results exposed in this work, whereas a putative perspective for the gene expression studies could be looking at which level is exerted the control of TRK1 gene.

Tipologia del documento
Tesi di dottorato
Pasquini, Miriam
Dottorato di ricerca
Settore disciplinare
Settore concorsuale
Parole chiave
Transketolase; thiamine-pyrophosphate (TPP); magnesium; Chlamydomonas reinhardtii; crystal structure; CD spectroscopy; enzyme activity assays
Data di discussione
18 Aprile 2017

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