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Issue 4, 2015
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Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson–Calvin cycle enzyme enslaved to its cysteine residues

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Abstract

Phosphoribulokinase (PRK) in the green alga Chlamydomonas reinhardtii is a finely regulated and well-studied enzyme of the Benson–Calvin cycle. PRK can form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small chloroplast protein CP12. This study aimed to determine the molecular determinants on PRK involved in the complex and the mechanism of action of a recently described novel regulation of PRK that involves glutathionylation. A combination of mass spectrometry, mutagenesis and activity analyses showed that Cys16, besides its role as the binding site of ATP, was also the site for S-glutathionylation. Previous kinetic analysis of the C55S mutant showed that in the oxidized inactive form of PRK, this residue formed a disulfide bridge with the Cys16 residue. This is the only bridge reported for PRK in the literature. Our data show for the first time that a disulfide bridge between Cys243 and Cys249 on PRK is required to form the PRK–GAPDH–CP12 complex. These results uncover a new mechanism for the PRK–GAPDH–CP12 formation involving a thiol disulfide exchange reaction with CP12 and identify Cys16 of PRK as a target of glutathionylation acting against oxidative stress. Although Cys16 is the key residue involved in binding ATP and acting as a defense against oxidative damage, the formation of the algal ternary complex requires the formation of another disulfide bridge on PRK involving Cys243 and Cys249.

Graphical abstract: Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson–Calvin cycle enzyme enslaved to its cysteine residues

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Article information


Submitted
13 Jan 2015
Accepted
06 Feb 2015
First published
06 Feb 2015

Mol. BioSyst., 2015,11, 1134-1145
Article type
Paper
Author version available

Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson–Calvin cycle enzyme enslaved to its cysteine residues

G. Thieulin-Pardo, T. Remy, S. Lignon, R. Lebrun and B. Gontero, Mol. BioSyst., 2015, 11, 1134
DOI: 10.1039/C5MB00035A

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