Pseudoreversion substitution at large-subunit residue 54 influences the CO2/O2 specificity of chloroplast ribulose-bisphosphate carboxylase/oxygenase.
Chlamydomonas reinhardtii mutant 31-4E lacks ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) holoenzyme due to a mutation in the chloroplast rbcL gene. This mutation causes a glycine54-to-aspartate substitution within the N-terminal domain of the Rubisco large subunit. In the present study, photosynthesis-competent revertants were selected to determine whether other amino acid substitutions might complement the primary defect. Revertants were found to arise from only true reversion or either of two forms of pseudoreversion affecting residue 54. One pseudorevertant has a glycine54-to-alanine substitution that decreases the accumulation of holoenzyme, but the purified Rubisco has near-normal kinetic properties. The other pseudorevertant has a glycine54-to-valine substitution that causes an even greater decrease in holoenzyme accumulation. Rubisco purified from this strain was found to have an 83% decrease in the Vmax of carboxylation and an 18% decrease in the CO2/O2 specificity factor. These results indicate that small increases in the size of amino acid side chains can influence Rubisco assembly or stability. Even though such changes occur far from the active site, they also play a significant role in determining Rubisco catalytic efficiency.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002245:Carbon Dioxide / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D017343:Genes, Plant; D005998:Glycine; D007700:Kinetics; D046911:Macromolecular Substances; D010100:Oxygen / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014633:Valine
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase; D014633:Valine; D010100:Oxygen; D005998:Glycine