Specificity for activase is changed by a Pro-89 to Arg substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.
Tobacco activase does not markedly facilitate the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1. 39) from non-Solanaceae species, including the green alga Chlamydomonas reinhardtii. To examine the basis of this specificity, we focused on two exposed residues in the large subunit of Rubisco that are unique to the Solanaceae proteins. By employing in vitro mutagenesis and chloroplast transformation, P89R and K356Q substitutions were separately made in the Chlamydomonas enzyme to change these residues to those present in tobacco. Both mutants were indistinguishable from the wild type when grown with minimal medium in the light and contained wild-type levels of holoenzyme. Purified Rubisco was assessed for facilitated activation by spinach and tobacco activase. Both wild-type and K356Q Rubisco were similar in that spinach activase was much more effective than tobacco activase. In contrast, P89R Rubisco was not activated by spinach activase but was well activated by tobacco activase. Thus, the relative specificities of the spinach and tobacco activases for Chlamydomonas Rubisco were switched by changing a single residue at position 89. This result provides evidence for a site on the Rubisco holoenzyme that interacts directly with Rubisco activase.
Larson
E M
EM
Department of Crop Sciences, University of Illinois, Urbana, Illinois 61801, USA.
O'Brien
C M
CM
Zhu
G
G
Spreitzer
R J
RJ
Portis
A R
AR
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii; D004789:Enzyme Activation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000378:metabolism*; D010947:Plants, Toxic; D010960:Plasminogen Activators / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D018724:Spinacia oleracea / Q000201:enzymology; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology
D010940:Plant Proteins; C054765:rca protein, plant; D001120:Arginine; D011392:Proline; D010960:Plasminogen Activators; D012273:Ribulose-Bisphosphate Carboxylase