Biochemistry, Department of


Date of this Version



Published in PNAS, November 22, 2005, vol. 102, no. 47, 17225–17230. Copyright © 2005 by The National Academy of Sciences of the USA. Used by Permission


Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO2 fixation and, thus, limits agricultural productivity. However, Rubisco enzymes from different species have different catalytic constants. If the structural basis for such differences were known, a rationale could be developed for genetically engineering an improved enzyme. Residues at the bottom of the large-subunit α/β-barrel active site of Rubisco from the green alga Chlamydomonas reinhardtii (methyl-Cys-256, Lys-258, and Ile-265) were previously changed through directed mutagenesis and chloroplast transformation to residues characteristic of land-plant Rubisco (Phe-256, Arg-258, and Val-265). The resultant enzyme has decreases in carboxylation efficiency and CO2/O2 specificity, despite the fact that land-plant Rubisco has greater specificity than the Chlamydomonas enzyme. Because the residues are close to a variable loop between β-strands A and B of the small subunit that can also affect catalysis, additional substitutions were created at this interface. When largesubunit Val-221 and Val-235 were changed to land-plant Cys-221 and Ile-235, they complemented the original substitutions and returned CO2/O2 specificity to the normal level. Further substitution with the shorter βA–βB loop of the spinach small subunit caused a 12–17% increase in specificity. The enhanced CO2/O2 specificity of the mutant enzyme is lower than that of the spinach enzyme, but the carboxylation and oxygenation kinetic constants are nearly indistinguishable from those of spinach and substantially different from those of Chlamydomonas Rubisco. Thus, this interface between large and small subunits, far from the active site, contributes significantly to the differences in catalytic properties between algal and land-plant Rubisco enzymes.