Lim, Boon Hoe , Spreitzer, Robert J. .
Phylogenetic engineering of ribulose-1,5-bisphosphate carboxylase/oxygenase.
The catalytic properties of 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. The green alga 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 Chlamydomonas 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. However, 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 can be 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 created and analyzed previously. In the present study, the remaining enzymes were created. All the mutant strains grow photosynthetically, and all but three of the mutant enzymes have normal catalytic properties. The G168P-L326I-M349L-M375L-A398S-C399V mutant 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 phylogenetic substitutions. This research is supported by the USDA NRI.
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University of Nebraska, Department of Biochemistry
1 - University of Nebraska, Department of Biochemistry, Lincoln, NE, 68588, USA
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM