Poster: Photosynthesis

Abs # 341: Altered carboxy-terminus/loop-6 interactions influence the catalytic efficiency and CO₂/O₂ specificity of Rubisco

Presenter:	Satagopan, Sriram , sriram@unlserve.unl.edu
Authors	Satagopan, Sriram  (A)   Spreitzer, Robert J (A)
Affiliations:	(A): University of Nebraska

Large-subunit loop 6 plays a critical role in discriminating between substrates CO₂ and O₂ at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Based on numerous X-ray crystal structures, this loop (Gly-333 through Gly-337) is either in a disordered (open) or ordered (closed) conformation. It has been proposed that invariant Asp-473 serves as a "latch" responsible for placing the large-subunit carboxy terminus (Trp-462 through Leu-475) over loop 6, thereby stabilizing the closed conformation that is required for catalysis. Taking advantage of the ease with which the Chlamydomonas chloroplast can be transformed, we used directed mutagenesis to create Asp-473-to-Ala (D473A) and Asp-473-to-Glu (D473E) mutant proteins in vivo. The D473A and D473E mutant strains are indistinguishable from wild type with respect to photoautotrophic growth, indicating that Asp-473 is not essential for catalysis. Furthermore, because the mutant proteins are present at wild-type levels in vivo and display wild-type thermal stability in vitro, the mutant substitutions do not appear to substantially alter holoenzyme structure. However, when the purified D473A and D473E enzymes were analyzed in detail, both were found to have a 2.5 fold increase in K_m(CO₂), 70% decrease in carboxylation V_max, and 15% decrease in CO₂/O₂ specificity. Asp-473 plays a critical role in Rubisco catalytic efficiency, but other interactions must be involved in keeping loop 6 in the closed conformation. Considering that substitutions at residue 473 can influence CO₂/O₂ specificity, further study of interactions between loop 6 and the carboxy terminus may provide clues for engineering an improved Rubisco. This work was supported by the USDA NRI.