Rubisco phylogenetic engineering
Boon Hoe Lim and Robert J. Spreitzer
Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664
The catalytic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) differ among divergent species. If the structural basis for this variation was known, it might be possible to identify targets for improving the enzyme. Chlamydomonas reinhardtii is amenable to both chloroplast (rbcL) and nuclear (rbcS) transformation. In a recent study, changing five large-subunit residues and a small-subunit loop to those of spinach Rubisco produced a mutant strain, named penta-ABSO, that has a Rubisco enzyme with increased CO2/O2 specificity and spinach-like catalytic properties. Phylogenetic analysis has identified 34 large-subunit residues that differ between Chlamydomonas and >95% of 500 land plants. To determine whether any of these other residues (in all combinations) may also contribute to catalytic variation would require the creation of 234 mutant enzymes. To simplify this problem, the phylogenetic residues were placed into 15 groups based on van der Waals contact in the highest-resolution Chlamydomonas Rubisco x-ray crystal structure. Five of these mutant enzymes were analyzed previously. In the present study, the remaining enzymes were created. The mutant strains grow photosynthetically, and all but three of the mutant enzymes have normal catalytic properties. The G168P-L326I-M349L-M375L-A398S-C399V enzyme has decreased CO2/O2 specificity, and the G442N-D443E-V444I-S447E and R305K-D470E-T471A-I472M-K474T enzymes have increased and decreased O2 inhibition of carboxylation, respectively. Because these phylogenetic residues must be complemented by other residues in land-plant Rubisco (which differ from those in Chlamydomonas), work is in progress to combine the groups of substitutions. However, despite being more like a plant enzyme, several of these new mutant enzymes lack function or fail to assemble. Therefore, work is also in progress to dissect the unique and beneficial residue substitutions in the penta-ABSO enzyme. This research is supported by the USDA NRI.
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