Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii [Enzymology]

September 8th, 2014 by Morisse, S., Michelet, L., Bedhomme, M., Marchand, C. H., Calvaresi, M., Trost, P., Fermani, S., Zaffagnini, M., Lemaire, S. D.

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well-established mechanism involved in the control of a large number of cellular processes including the Calvin-Benson cycle. Indeed, four out of eleven enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomic-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii (Cr) and we show that CrPGK1 activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (-335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates while the enzyme activation appeared specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses and 3D-modelling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys227 and Cys361. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzymes with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view.