In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop

Abstract

To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function.

Figure 1.

Biochemical properties of Rubisco activase isoforms from rice and agave. A, SDS-PAGE analysis of purified activases and rice Rubisco. A total of 4 µg of protein loaded per lane. B, Rubisco activation assays of RuBP-inhibited rice Rubisco (ER) performed at 25°C (0.5 µm Rubisco active sites, 5 µm activase protomer). C, ATPase activity assays (5 µm protomer, 25°C) and quantified Rubisco activase activity of the different activase isoforms. Error bars show mean and sd of independent experiments with n as indicated. Significant differences (P < 0.05) are denoted by different letters at the base of each bar.

Figure 2.

Agave activases are highly thermostable. A, The activase isoforms were incubated for 10 min at the indicated temperature, and subsequently the remaining ATPase activity was assayed at 25°C (5 µm protomer). B, ATPase activity of OsRcaβ and AtRcaβ at various temperatures. Proteins were preincubated (10 min) and subsequently measured at the indicated temperatures under the conditions described in Figure 1C. Error bars show mean and sd of independent experiments with n indicated on top of the bars. Significant differences between activities at the same temperature (P < 0.05) are denoted by different letters at the base of each bar. C, AtRcaβ has higher Rubisco activase activity than OsRcaβ even at low temperature. Rubisco activation assays were performed as in Figure 1B at 15°C. A and C, error bars indicate mean and sd of independent experiments (n = 3–5).

Figure 3.

Rice and agave activases form hetero-oligomers displaying intermediate functionality but subunit-specific thermostability. A and B, Rubisco activation assay (A) and ATPase assay (B) performed using 5 µm activase protomer (2.5 µm + 2.5 µm for AtRcaβ + OsRcaβ). ATPase is given per active site. C, Evidence for hetero-oligomerization. AtRcaβ was mixed with increasing amounts of ATPase inactive OsRcaβD173A, incubated for 10 min, and assayed for ATPase activity. D, Incubation at 45°C restores ATPase activity of impaired hetero-oligomers to levels of AtRcaβ wild type. C and D, ATPase activity was calculated as turnovers per wild-type active site. Error bars indicate mean and sd of independent experiments (A, n = 5–42; B and D, n = 4–7).

Figure 4.

Thermostability and high activase activity maps to residues 1 to 244 of AtRcaβ. A, Schematic representation of the chimeric activases. The crossover point corresponds approximately to the domain boundary of the nucleotide binding domain and the α-helical subdomain (residue 253 in OsRcaβ). Green, rice sequence; cyan, agave sequence. Numbering on top and bottom refers to residue numbers of the source sequence. B, The chimera containing the agave N-terminal portion (AtOsRcaβ) is thermostable. Thermostability assay performed as described in the legend to Figure 2A. Error bars indicate mean and sd of at least three independent experiments. C, The high activase activity of AtRcaβ maps to the N-terminal portion. Experimental conditions as in Figure 1C. Error bars show mean and sd of independent experiments with n = 3 to 4 (B) and as indicated on top of the bars (C). Significant differences (P < 0.05) are denoted by different letters at the base of each bar.

Figure 5.

The Glu-217-Gln substitution confers high in vitro activase activity to OsRcaβ. A, Sequence alignment of the thermostability-conferring region of rice and agave activases. Residues indicated by stars were selected for mutagenesis. B, Thermostability assay of the Rca variants. ATPase assays were performed at 25°C and after a 10-min preincubation at 45°C. C and D, OsRcaβE217Q phenocopies the high in vitro activase activity of AtRcaβ. Error bars indicate mean and sd of independent experiments (B, n = 3–7 at 25°C, n = 1 at 45°C; C as shown; D, n = 3–5).

Figure 6.

Characterization of a new Rubisco interacting loop. A, Relative position of the Q217-containing loop in context of the Rca hexamer. A surface representation of the hexameric tobacco Rca model (PDB:3ZW6) is shown with alternate subunits colored in blue and light blue. The specificity helix H9 is colored red, and the last resolved residues flanking the flexible loop are shown in orange (T206) and yellow (E218). The close-up view shows the local secondary structure with helices represented by cylinders. The loop is indicated by a dotted line. B, Logo motif of the loop from an alignment of 220 plant Rca sequences generated using WebLogo (Crooks et al., 2004). C, Biochemical characterization of loop mutants was performed as in Figure 1C. Error bars indicate mean and sd of independent experiments with n = 4 to 9. D, The loop region in the Arabidopsis Rca structure (PDB:4W5W) suggests that the carboxyl group of E217 forms a salt-bridge with the amino group of K162. For simplicity, residue numbering in all panels of this figure corresponds to the OsRcaβ sequence. Structures were drawn using Pymol (www.pymol.org).