Dataset Information

Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts.

ABSTRACT: In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent, measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges is simultaneous. In oxidized CP12, the C23-C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66-C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment: a cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66-C75 pair than for the C23-C31 pair. The redox mid-potentials for the two cysteine pairs differ and are similar to those found for glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase, consistent with the regulatory role of CP12.

SUBMITTER: Launay H

PROVIDER: S-EPMC8151241 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts.

Launay Helene H Shao Hui H Bornet Olivier O Cantrelle Francois-Xavier FX Lebrun Regine R Receveur-Brechot Veronique V Gontero Brigitte B

Biomolecules 20210508 5

In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent, measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed tha ...[more]

PMID: 34066751

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Project description:We report the first in vivo analysis of a canonical CP12 regulatory protein, namely the unique CP12 of the model cyanobacterium Synechocystis PCC 6803, which has the advantage of being able to grow photoautotrophically, photomixotrophically, and photoheterotrophically. The data showed that CP12 is dispensable to cell growth under standard (continuous) light and light/dark cycle, whereas it is essential for the catabolism of exogenously added glucose that normally sustains cell growth in absence of photosynthesis. Furthermore, to be active in glucose catabolism, CP12 requires its three conserved features: its AWD_VEEL motif and its two pairs of cysteine residues. Also interestingly, CP12 was found to regulate the redox equilibrium of NADPH, an activity involving its AWD_VEEL motif and its C-ter cysteine residues, but not its N-ter cysteine residues. This finding is important because NADPH powers up the methylerythritol 4-phosphate (MEP) pathway that synthesizes the geranyl-diphosphate (GPP) and farnesyl-diphosphate (FPP) metabolites, which can be transformed into high-value terpenes by recombinant cyanobacteria producing plant terpene synthase enzymes. Therefore, we have introduced into the Δcp12 mutant and the wild-type (control) strain our replicative plasmids directing the production of the monoterpene limonene and the sesquiterpene bisabolene. The photosynthetic production of both bisabolene and limonene appeared to be increased (more than two-fold) in the Δcp12 mutant as compared to the WT strain. Furthermore, the level of bisabolene production was also higher to those previously reported for various strains of Synechocystis PCC 6803 growing under standard (non-optimized) photoautotrophic conditions. Hence, the presently described Δcp12 strain with a healthy photoautotrophic growth and an increased capability to produce terpenes, is an attractive cell chassis for further gene manipulations aiming at engineering cyanobacteria for high-level photoproduction of terpenes.

Dataset Information

Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts.

Publications

Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts.

Similar Datasets

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