Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

Abstract

The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Towards that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N-terminus of the Rubisco large subunit. Large subunits lacking residues 1-4 are functional Rubiscos, but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model where Rca transiently threads the Rubisco large subunit N-terminus through the axial pore of the AAA+ hexamer.

The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos

“…Its mechanism involves an interaction with the Rubisco large subunit N-terminus, which is conserved in the higher plant system (41). The homologue encoded by Fremyella diplosiphon showed the expected carboxysomal localization(35).…”

An Insight of RuBisCO Evolution through a Multilevel Approach

“…Our results are in agreement with Watanabe et al [ 79 ] because the movements of the RbcL structure of G. partita presented a different residue-residue correlation pattern than IB and IC/D structures ( Figure 8 e). Although G. partita has more residues in the N-terminal and C-terminal regions ( Figure 3 ), this phenomenon could play an important role in the structural movement of the RuBisCO enzyme, as indicated by some studies [ 78 ]. Likewise, the RMSF analysis of 1IWA ( G. partita ) showed the greatest flexibility between residues ~Trp73–Ala86 (the connecting loop between αB and βC).…”

“…In addition, the N-terminal and C-terminal loops function like latches that hold loop ~64–85 and loop 6 in their closed positions with an extremely slow release of CABP [ 74 ]. Moreover, RuBisCO studies carried out on spinach and wheat were able to show that residues ~8 to 20 at the N-terminal end are only ordered when the active site is closed [ 78 ]. In the closed conformation, the N-terminal end (Phe13, Lys14, Gly16 and Lys18) is placed directly on the connecting loop between αB and βC (~64–85 residues), which coordinates the P1 site of the substrate [ 78 ].…”

“…Moreover, RuBisCO studies carried out on spinach and wheat were able to show that residues ~8 to 20 at the N-terminal end are only ordered when the active site is closed [ 78 ]. In the closed conformation, the N-terminal end (Phe13, Lys14, Gly16 and Lys18) is placed directly on the connecting loop between αB and βC (~64–85 residues), which coordinates the P1 site of the substrate [ 78 ]. In contrast, the open state is associated with weak products being attached (metal ions that are catalytically inert).…”

Characterization of Ribulose-1,5-Bisphosphate Carboxylase-Oxygenase Activase (Rca) Genes in Durum Wheat

“…Little is known about Rca detailed mechanism(s) in plants, since only recently plant Rubiscos have been recombinantly expressed (Ng et al 2020). Some speci c conditions might affect its e ciency, such as abiotic or biotic stresses, especially light, temperature and pathogen infection.…”