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bioRxiv · 10.1101/2022.09.29.510120

Structural and functional characterization of chloroplast ribulose-5-phosphate-3-epimerase from the model green microalga Chlamydomonas reinhardtii

Meloni, M.·Fanti, S.·Tedesco, D.·Gurrieri, L.·Trost, P.·Fermani, S.·Lemaire, S. D.·Zaffagnini, M.·Henri, J.
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Abstract

Photosynthetic carbon fixation relies on Rubisco and ten additional enzymes in the conserved Calvin-Benson-Bassham (CBB) cycle. Epimerization of xylulose-5-phosphate (X5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco activity. Ribulose-5-phosphate-3-epimerase (RPE) catalyzes the formation of Ru5P but it can also operate in the pentose phosphate pathway (PPP) by catalyzing the reverse reaction. Here, we describe the catalytic and structural properties of the recombinant form of photosynthetic RPE isoform 1 from Chlamydomonas reinhardtii (CrRPE1). The enzyme shows catalytic parameters that are variably comparable to those of the paralogues involved in the PPP and CBB cycle but with some notable exceptions. CrRPE1 is a homo-hexamer that exposes a catalytic pocket on the top of an 8{beta}8 triose isomerase-type (TIM-) barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. Despite being identified as a putative target of thiol-based redox modifications, CrRPE1 activity is not altered by redox treatments, indicating that the enzyme does not bear redox sensitive thiol groups and is not regulated by thiol-switching mechanisms. We mapped phosphorylation sites on the crystal structure and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. Overall, this work provides a detailed description of the catalytic and regulatory properties of CrRPE along with structural data, which allow for a deeper understanding of the functioning of this enzyme of the CBB cycle and in setting the basis for possible strategies to improve the photosynthetic metabolism.

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Meloni, M., Fanti, S., Tedesco, D., Gurrieri, L., Trost, P., Fermani, S., Lemaire, S. D., Zaffagnini, M., Henri, J.. 2022-09-30. Structural and functional characterization of chloroplast ribulose-5-phosphate-3-epimerase from the model green microalga Chlamydomonas reinhardtii. https://doi.org/10.1101/2022.09.29.510120

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