Minisymposium 12: Photosynthetic Carbon Metabolism
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M1202: Green-like small subunits with red-like loops assemble into the holoenzyme and affect catalysis of chloroplast Rubisco
During evolution, the greatest change in the structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has occurred in the nuclear-encoded small subunit. The loop between beta-strands A and B contains 22 and 28 residues in land plants and green algae, respectively, but only 10 residues in prokaryotes and nongreen eukaryotic algae. In some prokaryotes and all nongreen algae, a longer small-subunit carboxyl terminus forms a loop between two additional beta strands (E and F) that folds into the space above the shorter AB loop. The AB and EF loops of four small subunits surround each end of the central solvent channel that traverses the Rubisco holoenzyme. These small-subunit structures are 20 angstroms from the active sites that reside in the chloroplast-encoded large subunits. Some of the "red-like" (EF-loop-containing) Rubisco enzymes have CO2/O2 specificity more than twice as great as "green-like" Rubisco enzymes of land plants and green algae, and recent studies have shown that the small-subunit AB loop can influence large-subunit specificity. Therefore, we wondered whether the red-like small-subunit structures might increase Rubisco specificity. Because Rubisco enzymes with mutant small subunits can be recovered by transforming a mutant strain of the green alga Chlamydomonas reinhardtii, which lacks the small-subunit gene family, the AB and EF loops of the eukaryotic red alga Galdieria partita were engineered (separately and together) into the Chlamydomonas small subunit. Photosynthesis-competent transformants were recovered in every case. Although none of the chimeric-small-subunit mutants is temperature conditional for growth, all the purified mutant enzymes have decreased thermal stability in vitro. Detailed kinetic analysis of the purified enzymes revealed that the red-like Galdieria AB and EF loops individually cause decreases in carboxylation Vmax with no effect on CO2/O2 specificity. However, when combined together, the loops cause a 16% decrease in specificity due to even greater decreases in Vmax. Substantial decreases also occur in the Km CO2 and O2 values. Because the effects of the individual loops are not simply additive, it is likely that they interact in the AB/EF chimeric enzyme. The red-like small-subunit structures can influence large-subunit catalysis, but red-like large-subunit residues must also complement the small-subunit AB and EF loops to achieve a higher CO2/O2 specificity. This research is supported by the US DOE.
