PARENT SESSION

Symposium S3C C3 and Rubisco
Tuesday August 31st, 2004 10:20 AM-12:20 PM Room 510A
Chair: Michael Salvucci
Co-Chair: Akiho Yokota

Phylogenetic engineering of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in Chlamydomonas reinhardtii. Robert Spreitzer^*,1, Srinivasa Peddi¹, Sriram Satagopan¹, ¹ University of Nebraska, Lincoln, NE, USA

ABSTRACT- The ratio of carboxylation to oxygenation catalytic efficiencies (i. e., omega) varies by an order of magnitude among divergent Rubisco enzymes. Prokaryotes have the lowest values (15-50) whereas plants and nongreen algae have the highest (80-240). However, greater omega values are generally offset by lower carboxylation velocities, making it difficult to tell whether any Rubisco is better than any other in vivo. Nonetheless, an understanding of the molecular basis for catalytic diversity might be useful for engineering a better enzyme. The sequence of the active-site large subunit of Chlamydomonas Rubisco is 90% identical with the sequences of land plants, but the omega value (60) is 25% lower that that of land-plant Rubisco. When the Chlamydomonas large-subunit sequence is compared with the sequences of 500 land-plant species, only 34 residues are seen to be unique to Chlamydomonas (present in <5% of plants). However, to change each of these phylogenetic residues to the plant residue, one at a time in all possible combinations, would require the creation and analysis of 2³⁴ enzymes. Because mutant screening and selection in Chlamydomonas have already identified residues that influence omega, we focused on those regions. Whereas two directed-mutant substitutions in the large-subunit N-terminal domain had only minor influence over catalysis, three large-subunit phylogenetic substitutions at an interface with small subunits caused a substantial decrease in omega and carboxylation efficiency. These three residues must be complemented in land plants by other residues that differ from those of Chlamydomonas. Introduction of two more large-subunit phylogenetic substitutions returned omega to the wild-type value, and addition of a loop of plant small-subunit residues increased omega by 12% relative to the wild-type value. Examination of ten phylogenetic substitutions in the region surrounding active-site loop 6 has revealed that four substitutions in the large-subunit C terminus also produce a 12% increase in omega. Although neither of these higher-omega enzymes is improved with respect to other catalytic properties, the regions identified by phylogenetic engineering warrant further attention as we contemplate the design of an improved Rubisco. Research was supported by grants from the USDA NRI and US DOE.

KEY WORDS: Chlamydomonas, ribulose-1,5-bisphosphate carboxylase/oxygenase, chloroplast, evolution