Journal
MOLECULAR BIOSYSTEMS
Volume 11, Issue 4, Pages 1134-1145Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c5mb00035a
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Funding
- Centre National de la Recherche Scientifique (CNRS)
- Aix-Marseille Universite, La Region PACA, APO project [063 506]
- Ecole Normale Superieure, Cachan
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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.
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