Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii

Background In photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg2 +. Methods TK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography. Results CrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg2 +. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg2 +-dependent and proceeds with a slow kinetics. The 3Dâstructure of CrTK without cofactors (CrTKapo) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg2 +participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation. Conclusion These evidences indicate that Mg2 +is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg2 +is involved in the control of redox sensitivity of CrTK. General significance The importance of Mg2 +in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg2 +in chloroplasts

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

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