title: Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde-3-phosphate dehydrogenase in the assembly process of a bi-enzyme complex involved in CO2 assimilation.
abstract: The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the chloroplast of Chlamydomonas reinhardtii is part of a complex that also includes phosphoribulokinase (PRK) and CP12. We identified two residues of GAPDH involved in protein-protein interactions in this complex, by changing residues K128 and R197 into A or E. K128A/E mutants had a Km for NADH that was twice that of the wild type and a lower catalytic constant, whatever the cofactor. The kinetics of the mutant R197A were similar to those of the wild type, while the R197E mutant had a lower catalytic constant with NADPH. Only small structural changes near the mutation may have caused these differences, since circular dichroism and fluorescence spectra were similar to those of wild-type GAPDH. Molecular modelling of the mutants led to the same conclusion. All mutants, except R197E, reconstituted the GAPDH-CP12 subcomplex. Although the dissociation constants measured by surface plasmon resonance were 10-70-fold higher with the mutants than with wild-type GAPDH and CP12, they remained low. For the R197E mutation, we calculated a 4 kcal/mol destabilizing effect, which may correspond to the loss of the stabilizing effect of a salt bridge for the interaction between GAPDH and CP12. All the mutant GAPDH-CP12 subcomplexes failed to interact with PRK and to form the native complex. The absence of kinetic changes of all the mutant GAPDH-CP12 subcomplexes, compared to wild-type GAPDH-CP12, suggests that mutants do not undergo the conformation change essential for PRK binding.
authors.affiliation: Laboratoire Génétique et Membranes, Département Biologie Cellulaire, Institut Jacques Monod, UMR 7592 CNRS, Universités Paris VI-VII, Paris, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Graciet
authors.forename: Guillermo
authors.initials: G
authors.lastname: Mulliert
authors.forename: Sandrine
authors.initials: S
authors.lastname: Lebreton
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gontero
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism*; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D002245:Carbon Dioxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase