Several studies identified the Calvin–Benson cycle (CBC), the photosynthetic pathway responsible for carbon fixation (Figure 1), as a redox-regulated process. New biochemical and proteomic approaches suggest that all the CBC enzymes may withstand redox regulation through multiple redox post- translational modifications (PTMs), like disulfide bond formation, glutathionylation and nitrosylation 1 . This redox control is likely involved in the adaptative response to environmental conditions, including light/dark transitions and abiotic/biotic stress. This ongoing work aims at investigating the redox regulation of the chloroplastic CBC enzyme transketolase from the green microalga Chlamydomonas reinhardtii (CrTK). Recombinant CrTK was heterologously expressed in E. coli and purified to homogeneity through metal affinity chromatography. In vitro activity assays showed that the purified enzyme displays a redox-sensitivity being partially inhibited by oxidizing treatments. Moreover, the 3D-structure of the reduced protein (solved at 1.8 Å resolution) revealed the presence of several cysteine couples located at suitable distance for disulfide bond formation and in close proximity to catalytic residues. The influence of the redox state on the structural features of CrTK was further investigated by LC-ESI-MS/MS analysis, allowing the identification of the regulatory cysteines. Concomitantly, circular dichroism (CD) analysis in the far-UV spectral region allowed evaluating redox-dependent changes in the secondary structure of the enzyme. Furthermore, CD analysis in the near-UV region showed the onset of an induced circular dichroism (ICD) signal 3 arising from the interaction between CrTK and the cofactor thiamine pyrophosphate (TPP), which was exploited to monitor CrTK-TPP binding under different redox states of the enzyme.

Daniele Tedesco, M.N. (2016). Redox-sensitive structural features of transketolase from Chlamydomonas reinhardtii (CrTK).

Redox-sensitive structural features of transketolase from Chlamydomonas reinhardtii (CrTK)

Daniele Tedesco
;
Marina Naldi;PASQUINI, MIRIAM;Mirko Zaffagnini;Francesco Francia;Carlo Bertucci
2016

Abstract

Several studies identified the Calvin–Benson cycle (CBC), the photosynthetic pathway responsible for carbon fixation (Figure 1), as a redox-regulated process. New biochemical and proteomic approaches suggest that all the CBC enzymes may withstand redox regulation through multiple redox post- translational modifications (PTMs), like disulfide bond formation, glutathionylation and nitrosylation 1 . This redox control is likely involved in the adaptative response to environmental conditions, including light/dark transitions and abiotic/biotic stress. This ongoing work aims at investigating the redox regulation of the chloroplastic CBC enzyme transketolase from the green microalga Chlamydomonas reinhardtii (CrTK). Recombinant CrTK was heterologously expressed in E. coli and purified to homogeneity through metal affinity chromatography. In vitro activity assays showed that the purified enzyme displays a redox-sensitivity being partially inhibited by oxidizing treatments. Moreover, the 3D-structure of the reduced protein (solved at 1.8 Å resolution) revealed the presence of several cysteine couples located at suitable distance for disulfide bond formation and in close proximity to catalytic residues. The influence of the redox state on the structural features of CrTK was further investigated by LC-ESI-MS/MS analysis, allowing the identification of the regulatory cysteines. Concomitantly, circular dichroism (CD) analysis in the far-UV spectral region allowed evaluating redox-dependent changes in the secondary structure of the enzyme. Furthermore, CD analysis in the near-UV region showed the onset of an induced circular dichroism (ICD) signal 3 arising from the interaction between CrTK and the cofactor thiamine pyrophosphate (TPP), which was exploited to monitor CrTK-TPP binding under different redox states of the enzyme.
2016
XXIV National Meeting on Medicinal Chemistry (NMMC 2016)
Daniele Tedesco, M.N. (2016). Redox-sensitive structural features of transketolase from Chlamydomonas reinhardtii (CrTK).
Daniele Tedesco, Marina Naldi, Miriam Pasquini, Mirko Zaffagnini, Francesco Francia, Carlo Bertucci
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/640289
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