Several studies identified the Calvin\u2013Benson 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 \uc5
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
