Phylogenetic engineering of ribulose -1,5 -bisphosphate carboxylase /oxygenase large-subunit loop 6 in Chlamydomonas reinhardtii

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO2 fixation. Competitive inhibition by O2 and the loss of fixed carbon through oxygenase activity limit photosynthetic productivity. Consequently, Rubisco has been viewed as a potential target for improving catalytic efficiency and CO 2/O2 specificity as a means for improving agricultural productivity. However, it has been difficult to deduce ways to improve the enzyme because the active-site residues are nearly 100% conserved. Nonetheless, Rubisco enzymes from different species have different catalytic properties, and with a greater understanding of the structural basis for these differences, it may be possible to engineer a “better” Rubisco. In a previous study, the comparison of large-subunit sequences from ∼500 land-plants and the green alga Chlamydomonas reinhardtii identified a small set of residues that differed in regions previously shown by mutant screening to influence CO2/O2 specificity. A loop-6 amino-acid substitution, V331A, was complemented by a T342I or G344S substitution. In the present study, classical genetics, directed mutagenesis, and structural analysis identified additional residues in the loop-6 region and carboxy terminus that influence CO2/O2 specificity, indicating that subtle interactions in the loop-6 region and the carboxy terminus may be important determinants of catalytic efficiency. Ten residues in the loop-6 region differ between Chlamydomonas and land plants. Because it is difficult to analyze 10 substitutions in all possible combinations, these were combined into three groups based on their location in the X-ray crystal structure of Chlamydomonas Rubisco. Whereas loop-6 (L326I/V341I/M349L) and base-of-loop-6 (M375L/A398S/C399V) substitutions had little effect on catalytic properties, the carboxy terminal substitutions (D470P/T471A/I472M/K474T) caused a small but significant increase in CO2/O2 specificity. Despite this positive change, the other kinetic properties of the carboxy-terminal mutant enzyme remain quite different from the land-plant enzyme. Combining all 10 substitutions resulted in the loss of Rubisco holoenzyme in vivo. Thus, there must be additional residues that differ between Chlamydomonas and land plants that complement the 10 substitutions. These divergent and more-distant residues may account for differences in catalytic properties