Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) 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 alpha/beta-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 CO(2)/O(2) 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 beta-strands A and B of the small subunit that can also affect catalysis, additional substitutions were created at this interface. When large-subunit Val-221 and Val-235 were changed to land-plant Cys-221 and Ile-235, they complemented the original substitutions and returned CO(2)/O(2) specificity to the normal level. Further substitution with the shorter betaA-betaB loop of the spinach small subunit caused a 12-17% increase in specificity. The enhanced CO(2)/O(2) 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.

Fig. 1.

Stereo images of large-subunit phylogenetic (in black) or mutant/suppressor (in gray) residues that surround the small-subunit βA-βB loop (in white) in the x-ray crystal structures of Chlamydomonas (A) and spinach (B) Rubisco (1GK8 and 8RUC, respectively) (15, 16). The central solvent channel of the holoenzyme is in front of the displayed structures. Only large-subunit phylogenetic residues 221, 235, 256, 258, and 265 differ between Chlamydomonas and spinach Rubisco in this region (14). In Chlamydomonas, an L290F substitution caused a decrease in Ω (17), and suppressor substitutions in the large subunit (A222T or V262L) or small subunit (N54S or A57V, colored gray in only the Chlamydomonas structure) returned Ω to the normal value (18-20). The small-subunit βA-βB loop contains 28 residues in Chlamydomonas and 22 residues in spinach Rubisco (7).

Fig. 2.

Spot tests to assess the photoautotrophic growth of wild type (spot 1), large-subunit triple-mutant C256F/K258R/I265V (spot 2), large-subunit pentamutant V221C/V235I/C256F/K258R/I265V (spot 3), and penta/ABSO-mutant V221C/V235I/C256F/K258R/I265V/ABSO (spot 4), which contains the small-subunit βA-βB loop of spinach (13). Equal numbers of dark-grown cells were plated on minimal medium in the light (80 μmol of photons per m²/sec), at either the normal growth temperature of 25°C or elevated temperature of 35°C.

Fig. 3.

Western blot analysis of total soluble proteins from wild type (lanes 1 and 6), large-subunit triple-mutant C256F/K258R/I265V (lanes 2 and 7), small-subunit chimeric-mutant ABSO (lanes 3 and 8) (13), large-subunit pentamutant V221C/V235I/C256F/K258R/I265V (lanes 4 and 9), and penta/ABSO-mutant V221C/V235I/C256F/K258R/I265V/ABSO (lanes 5 and 10). Extracts (30 μg per lane) of cells grown at either 25°C (lanes 1-4) or 35°C (lanes 6-10) in darkness were fractionated by SDS/PAGE (7.5-15%) (30, 31). The Rubisco large (LS) and small (SS) subunits were detected with anti-Chlamydomonas Rubisco IgG (31).

Fig. 4.

Thermal inactivation of purified Rubisco from wild type (○), large-subunit triple-mutant C256F/K258R/I265V (•), small-subunit chimeric-mutant ABSO (□) (13), large-subunit pentamutant V221C/V235I/C256F/K258R/I265V (▪), and penta/ABSO-mutant V221C/V235I/C256F/K258R/I265V/ABSO (▵). Rubisco was incubated at each temperature for 10 min, cooled on ice, and assayed for RuBP carboxylase activity at 25°C (36). Activities were normalized to the specific activities measured after the 35°C incubation. Illustrated values did not differ by more than 10% of maximal activities in three independent experiments with separate enzyme preparations.

Fig. 5.

Interactions of large-subunit phylogenetic residues 258 (A and B), 256 (C and D), and 235 (E and F) in the x-ray crystal structures of Rubisco from Chlamydomonas (1GK8) (13), spinach (8RUC) (10), and Chlamydomonas chimeric-mutant ABSO (1UZD), which contains the small-subunit βA-βB loop of spinach (13). The Chlamydomonas structure (in white) is aligned with the structures (in dark gray) of spinach (A, C, and E) or ABSO (B, D, and F). The central solvent channel of the holoenzyme is behind all of the displayed structures.