Chimeric Small Subunits Influence Catalysis without Causing Global Conformational Changes in the Crystal Structure of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

Comparison of subunit sequences and X-ray crystal structures of ribulose-1,5-bisphosphate carboxylase/oxygenase indicates that the loop between β-strands A and B of the small subunit is one of the most variable regions of the holoenzyme. In prokaryotes and nongreen algae, the loop contains 10 residues. In land plants and green algae, the loop is comprised of ∼22 and 28 residues, respectively. Previous studies indicated that the longer βA−βB loop was required for the assembly of cyanobacterial small subunits with plant large subunits in isolated chloroplasts. In the present study, chimeric small subunits were constructed by replacing the loop of the green alga Chlamydomonas reinhardtii with the 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 loop size 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 βA−βB loops and the respective loops in the Synechococcus and spinach enzymes. The side chains of several large-subunit residues are altered in regions previously shown by directed mutagenesis to influence CO2/O2 specificity. Differences in the catalytic properties of divergent Rubisco enzymes may arise from differences in the small-subunit βA−βB loop. This loop may be a worthwhile target for genetic engineering aimed at improving photosynthetic CO2 fixation.