C172S substitution in the chloroplast-encoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase.
Previous work has indicated that the turnover of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1. 39) may be controlled by the redox state of certain cysteine residues. To test this hypothesis, directed mutagenesis and chloroplast transformation were employed to create a C172S substitution in the Rubisco large subunit of the green alga Chlamydomonas reinhardtii. The C172S mutant strain was not substantially different from the wild type with respect to growth rate, and the purified mutant enzyme had a normal circular dichroism spectrum. However, the mutant enzyme was inactivated faster than the wild-type enzyme at 40 and 50 degrees C. In contrast, C172S mutant Rubisco was more resistant to sodium arsenite, which reacts with vicinal dithiols. The effect of arsenite may be directed to the cysteine 172/192 pair that is present in the wild-type enzyme, but absent in the mutant enzyme. The mutant enzyme was also more resistant to proteinase K in vitro at low redox potential. Furthermore, oxidative (hydrogen peroxide) or osmotic (mannitol) stress-induced degradation of Rubisco in vivo was delayed in C172S mutant cells relative to wild-type cells. Thus, cysteine residues could play a role in regulating the degradation of Rubisco under in vivo stress conditions.
Moreno
J
J
Departament de Bioquímica i Biologia Molecular, Universitat de València, Burjassot E-46100, Spain. joaquin.moreno@uv.es
Spreitzer
R J
RJ
D019943:Amino Acid Substitution; D000818:Animals; D016825:Chlamydomonas reinhardtii; D002736:Chloroplasts / Q000378:metabolism*; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010641:Phenotype; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D012694:Serine; D012273:Ribulose-Bisphosphate Carboxylase; D003545:Cysteine