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

Accepted Manuscript version. The Published Journal Article is available on Biochimica et Biophysica Acta (BBA) – General Subjects, Volume 1861, Issue 8, Pages 2132–2145 (DOI: https://doi.org/10.1016/j.bbagen.2017.05.021). Supplementary Material available free of charge on the article webpage.

© 2017. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/

ABSTRACT

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 3D-structure of CrTK without cofactors (CrTK_apo) 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.