title: Thioredoxin-dependent redox regulation of chloroplastic phosphoglycerate kinase from Chlamydomonas reinhardtii.
abstract: 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, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-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, and we show that C. reinhardtii PGK1 (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, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys(227) and Cys(361). 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 enzyme 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.
authors.affiliation: From CNRS, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France, the Sorbonne Universités, Université Pierre et Marie Curie (UPMC) Universit́ Paris 06, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Samuel
authors.initials: S
authors.lastname: Morisse
authors.affiliation: From CNRS, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France, the Sorbonne Universités, Université Pierre et Marie Curie (UPMC) Universit́ Paris 06, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Laure
authors.initials: L
authors.lastname: Michelet
authors.affiliation: From CNRS, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France, the Sorbonne Universités, Université Pierre et Marie Curie (UPMC) Universit́ Paris 06, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Mariette
authors.initials: M
authors.lastname: Bedhomme
authors.affiliation: From CNRS, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France, the Sorbonne Universités, Université Pierre et Marie Curie (UPMC) Universit́ Paris 06, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Christophe H
authors.initials: CH
authors.lastname: Marchand
authors.affiliation: the Department of Chemistry "G. Ciamician," University of Bologna, 40126 Bologna, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Calvaresi
authors.affiliation: the Laboratory of Plant Redox Biology, Department of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy, and.
authors.forename: Paolo
authors.initials: P
authors.lastname: Trost
authors.affiliation: the Department of Chemistry "G. Ciamician," University of Bologna, 40126 Bologna, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Fermani
authors.affiliation: the Laboratory of Plant Redox Biology, Department of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy, and mirko.zaffagnini3@unibo.it.
authors.forename: Mirko
authors.initials: M
authors.lastname: Zaffagnini
authors.affiliation: From CNRS, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France, the Sorbonne Universités, Université Pierre et Marie Curie (UPMC) Universit́ Paris 06, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France, stephane.lemaire@ibpc.fr.
authors.forename: Stéphane D
authors.initials: SD
authors.lastname: Lemaire
mesh_terms: D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects; D054479:Chloroplast Thioredoxins / Q000378:metabolism*; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects / Q000528:radiation effects; D010449:Peptide Mapping; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D034421:Sus scrofa
chemical_list: D054479:Chloroplast Thioredoxins; D004220:Disulfides; D050505:Mutant Proteins; D010735:Phosphoglycerate Kinase; D003545:Cysteine; D004229:Dithiothreitol
keywords: Calvin-Benson Cycle; Chlamydomonas; Chloroplast; Disulfide; Phosphoglycerate Kinase; Photosynthesis; Redox Signaling; Thiol; Thioredoxin