Genetic Modification of Rubisco in Chlamydomonas: The chloroplast enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes either the carboxylation or oxygenation of RuBP. The relative efficiencies of these two reactions determine net carbon dioxide fixation in photosynthesis. A significant increase in plant productivity may be realized by increasing the ratio of carboxylase/oxygenase activities. Since the catalytic site of the enzyme is located on the chloroplast-encoded large-subunit polypetide, photosynthesis-deficient chloroplast mutants of the green alga Chlamydomonas reinhardii will be analyzed for altered enzyme specificity. Detailed biochemical analysis of Rubisco enzymes from these mutants will be correlated with the primary structure of the mutant large-subunit protein; mutant genes will be cloned and subjected to DNA sequencing. Photosynthesis-competent revertants will be selected from the mutants, and Rubisco will be analyzed as described for mutants. The proposed work is significant for several reasons. First, Chlamydomonas reinhardii has a Rubisco that is similar to the enzyme in higher plant. Beneficial changes identified in the proposed project could be engineered into other plants. Second, C. reinhardii is the only organism in which chloroplast mutations can be induced and recovered on a routine basis. Third, mutations that cause only a partial deficiency of Rubisco function are being recovered randomly. Thus, structural determinants of enzyme function may be identified that have not been identified by previous biochemical or molecular approaches. Fourth, revertants can be selected for restored Rubisco function and an elevated carboxylase/oxygenase ratio. Complementing mutations will define the relationships between three-dimensional enzyme structure and Rubisco function. In conclusion, the project will combine biochemistry, molecular biology and genetics to generate new information about the structure/function relationships of Rubisco.
