PARENT SESSION
Posters P3C C3 and Rubisco. Abstracts (617-630)

Evolutionary divergence in the structure of the Rubisco small-subunit betaA-betaB loop is not essential for assembly but influences large-subunit catalysis. Srinivasa Peddi¹, Saeid Karkehabadi², Md. Anwaruzzaman¹, Inger Andersson², Robert Spreitzer^*,1, ¹ University of Nebraska, Lincoln, NE, USA² Swedish University of Agricultural Sciences, Uppsala, Sweden

ABSTRACT- When subunit sequences and x-ray crystal structures of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are compared among diverse species, the loop between beta strands A and B of the small subunit is seen to be one of the most variable regions of the holoenzyme. In prokaryotes and nongreen algae, the loop contains only 10 residues. In land plants and green algae, the loop is comprised of 22 and 28 residues, respectively. Previous studies indicated that the longer betaA-betaB loop was required for the assembly of cyanobacterial small subunits with plant large subunits in isolated chloroplasts. However, by exploiting genetic methods in the green alga Chlamydomonas reinhardtii, it was shown that substitutions in the betaA-betaB loop could also influence carboxylation catalytic efficiency and CO₂/O₂ specificity despite being 20 angstroms away from the large-subunit active site. In the present study, chimeric small subunits were constructed in which the betaA-betaB loop of Chlamydomonas was replaced precisely by the loop sequences of Synechococcus or spinach. When these engineered genes were transformed into a Chlamydomonas mutant that lacks small-subunit genes, photosynthesis-competent colonies were recovered, indicating that the size of the loop is not essential for holoenzyme assembly. Whereas the Synechococcus loop causes decreases in carboxylation V_max, K_m(O₂), and CO₂/O₂ specificity, the spinach loop causes complementary decreases in carboxylation V_max, K_m(O₂), and K_m(CO₂) without a change in specificity. X-ray crystal structures of the engineered proteins reveal remarkable similarity between the introduced betaA-betaB loops and the respective loops in the crystal structures of Synechococcus and spinach Rubisco. There are subtle alterations in a number of large-subunit residues that contact the loops, several of which occur in regions previously shown by directed mutagenesis to influence catalytic efficiency and specificity. In conclusion, differences in catalytic properties among divergent Rubisco enzymes may arise from divergence of the small-subunit betaA-betaB loop. This loop may be a worthwhile target for genetic engineering aimed at improving Rubisco catalysis. Research was supported by grants from the U. S. Department of Energy and Swedish Research Council (FORMAS).

KEY WORDS: ribulose-1,5-bisphosphate carboxylase/oxygenase, Chlamydomonas, x-ray crystal structure, catalysis