DETERMINING THE EFFECT OF SUBSTITUTIONS AT ALANINE 47 IN SYNECHOCOCCUS PCC6301 RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO)
Mutant screening and genetic selection in various organisms have shown that residues far from the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) can influence catalytic efficiency and CO2/O2 specificity. Because RubisCO catalyzes the rate-limiting step of photosynthesis, further study of these sites distant from the primary reaction center may provide the necessary information for engineering an increase in primary productivity of crop plants. In a previously described system of random mutagenesis and bioselection (Smith, 2002), the RubisCO genes from the cyanobacterium Synechococcus PCC6301, were randomly mutated and introduced into the photosynthetic bacterium Rhodobacter capsulatus. An A47T substitution resulted in a very modest loss of specific activity. However, this mutant was not functional relative to the wild-type when complementation was performed. Since mutants with even lower catalytic activity were able to complement a RubisCO deletion strain to photoautotrophic growth, it was hypothesized that the A47T mutation affected important kinetic properties of RubisCO. To further explore the role of this residue, A47 in the cyanobacterial enzyme was changed to Glycine and Proline by site-directed mutagenesis. Analysis of the recombinant proteins demonstrated that both mutant enzymes exhibited lower specific activity than the wild-type enzyme when expressed in E. coli. To examine the possibility that the decrease in specific activity was due to decreased expression of the mutant enzymes, SDS-PAGE and Western Blots were performed. SDS-PAGE and Western blotting indicated that there was slightly less of the enzyme present for the mutants. This could be the result of decreased synthesis or increased degradation. Native PAGE demonstrated that the G mutant might be a folding-mutant, since it does not have any detectable holoenzyme on non-denaturing PAGE. To further support this hypothesis, there was also a doublet band present in the mutants on non-denaturing PAGE that was not present in the wild-type. Perhaps this additional band is also an indicator of a misfolded large subunit or an indicator that the polypeptides are closely related in structure. However, the proline mutant was able to form a holoenzyme on non-denaturing PAGE. The results confirm that A47 plays a critical role in the function of RubisCO.
School:Wright State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:rubisco photosynthesis calvin cycle plants synechococcus pcc6301 carbon fixation
Date of Publication:01/01/2006