Glycine 176 affects catalytic properties and stability of the Synechococcus sp. strain PCC6301 ribulose-1,5-bisphosphate carboxylase/oxygenase.
A previously described system for biological selection of randomly mutagenized ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) employing the phototrophic bacterium Rhodobacter capsulatus was used to select a catalytically altered form of a cyanobacterial (Synechococcus sp. strain PCC6301) enzyme. This mutant Rubisco, in which conserved glycine 176 was replaced with an aspartate residue, was not able to support CO(2)-dependent growth of the host strain. Site-directed mutant proteins were also constructed, e.g. asparagine and alanine residues replaced the native glycine with the result that these mutant proteins either greatly reduced the ability of R. capsulatus to support growth or had little effect, respectively. Growth phenotypes were consistent with the Rubisco activity levels associated with these proteins, and this was also borne out with purified recombinant proteins. Despite being catalytically challenged, the G176D and G176N mutant proteins were found to exhibit a more favorable interaction with CO(2) than the wild type protein but exhibited a reduced affinity for the substrate ribulose 1,5-bisphosphate. The G176A enzyme differed little from the wild type protein in these properties. None of the mutants had CO(2)/O(2) specificities that differed markedly from the wild type. Further studies taken from the known structure of the Synechococcus Rubisco indicated that substitutions at Gly-176 affected associations between large subunits. Supporting experimental data included an unusual protein concentration-dependent effect on in vitro activity, differences in thermal stability relative to the wild type protein, and aberrant migration on nondenaturing polyacrylamide gels. From these results, it is apparent that residues not directly located within the active site but near large subunit interfaces can affect key kinetic properties of Rubisco. These results suggest that further bioselection protocols (using these proteins as starting material) might yield novel mutant forms of Rubisco that relate to key functional properties.
Smith
Stephanie A
SA
Department of Microbiology and Plant Molecular Biology/Biotechnology Program, The Ohio State University, Columbus, Ohio 43210-1292, USA.
Tabita
F Robert
FR
D002384:Catalysis; D000458:Cyanobacteria / Q000201:enzymology* / Q000254:growth & development; D005998:Glycine; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000378:metabolism
D012273:Ribulose-Bisphosphate Carboxylase; D005998:Glycine