The chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO2 fixation, and thus limits plant productivity. Oxygenation of substrate ribulosebisphosphate leads to the loss of fixed carbon, and the competition of O2 with CO2 at the enzyme active site reduces the maximum rate of carboxylation. Genetic engineering is expected to improve carboxylation by changing the CO2/O2 specificity of the enzyme. However, there has been no rationale for accomplishing this goal. Directed mutagenesis has so far been limited to confirming the essential role of a variety of "active-site" residues, and the Rubisco primary and x-ray-crystal structures have failed to identify potential sites for beneficial modification. In contrast, the neoclassical genetic approach that can be pursued in the green alga Chlamydomonas reinhardtii has been able to define structural regions of the chloroplast-encoded large subunit that influence the CO2/O2 specificity of the holoenzyme. A more methodical approach for the application of induced mutations is underway. Mutations are being defined by sequencing appropriate chloroplast or nuclear genes, and mutant enzymes are being characterized biochemically to correlate changes in structure with changes in function. Directed mutagenesis and chloroplast transformation are also being used to test hypotheses formulated from the study of these mutations. The photosynthetic enzyme Rubisco is present in all green plants and algae and is considered to be the most abundant protein in nature. Since it catalyzes a reaction which can limit photosynthetic CO2 fixation and, therefore, plant growth, the long-term objective of this work is to make a better Rubisco. Since the primary structure of the Chlamydomonas reinhardtii Rubisco is quite similar to that of plants, it is a good model for defining potential modifications in crop-plant Rubisco enzymes.
