The Diverse AAA+ Machines that Repair Inhibited Rubisco Active Sites

Abstract

Gaseous carbon dioxide enters the biosphere almost exclusively via the active site of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). This highly conserved catalyst has an almost universal propensity to non-productively interact with its substrate ribulose 1,5-bisphosphate, leading to the formation of dead-end inhibited complexes. In diverse autotrophic organisms this tendency has been counteracted by the recruitment of dedicated AAA+ (ATPases associated with various cellular activities) proteins that all use the energy of ATP hydrolysis to remodel inhibited Rubisco active sites leading to release of the inhibitor. Three evolutionarily distinct classes of these Rubisco activases (Rcas) have been discovered so far. Green and red-type Rca are mostly found in photosynthetic eukaryotes of the green and red plastid lineage respectively, whereas CbbQO is associated with chemoautotrophic bacteria. Ongoing mechanistic studies are elucidating how the various motors are utilizing both similar and contrasting strategies to ultimately perform their common function of cracking the inhibited Rubisco active site. The best studied mechanism utilized by red-type Rca appears to involve transient threading of the Rubisco large subunit C-terminal peptide, reminiscent of the action performed by Clp proteases. As well as providing a fascinating example of convergent molecular evolution, Rca proteins can be considered promising crop-improvement targets. Approaches aiming to replace Rubisco in plants with improved enzymes will need to ensure the presence of a compatible Rca protein. The thermolability of the Rca protein found in crop plants provides an opportunity to fortify photosynthesis against high temperature stress. Photosynthesis also appears to be limited by Rca when light conditions are fluctuating. Synthetic biology strategies aiming to enhance the autotrophic CO₂ fixation machinery will need to take into consideration the requirement for Rubisco activases as well as their properties.

Keywords: AAA+ proteins; Rubisco; activase; carbon fixation; molecular chaperones; photosynthesis.

Figure 1

Rubisco's reaction mechanism and its inhibition properties. (A) A complex conserved reaction mechanism evolved to carboxylate ribulose 1,5-bisphosphate. The enediol intermediate can react with both oxygen and carbon dioxide. If oxygenation occurs the toxic metabolite 2-phosphoglycolate (2PG) is generated, which must be subjected to metabolite repair. (B) To perform the carboxylase reaction a conserved active site lysine (Lys-201 in spinach RbcL) must react with a non-substrate CO₂ to form a carbamate (EC), followed by the binding of a Mg²⁺ ion to form the catalytically competent holoenzyme ECM. (C) Both the inactive apo (E) and the active holoenzyme (ECM) are prone to dead-end inhibition by sugar phosphates such as RuBP, which binds to E and CA1P (2-carboxy-D-arabinitol 1-phosphate), which binds to ECM. Rubisco activases (Rca) recognize inhibited active sites and use the energy of ATP hydrolysis to cause a conformational change that releases the inhibitor.

Figure 2

Hypothetical scheme for the evolution of Rubisco and its activases. Following the great oxidation event at least three different classes of Rubisco activase were recruited from the general molecular chaperone machinery toward a specialized Rubisco activase function in diverse autotrophic organisms. Green type and red-type Rca was maintained in eukaryotic phototrophs of the green and the red plastid lineage respectively. A phylogenetic tree was drawn using Rubisco large subunit sequences that are associated with activases. It is important to note that regarding non-red prokaryotic Rubisco sequences, many instances exist that do not have identifiable activase genes encoded in the same genome. Surface representations of a hexameric Form II Rubisco (pdb:4lf1) and spinach Form I Rubisco (pdb:8ruc) are shown. Structures shown in this paper were drawn using pymol.

Figure 3

Structural features of inhibited Rubisco complexes. A comparison of structural elements involved in the Rca-mediated activation of Form I (A) and Form II (B) Rubisco. Left panels: Surface representation of CABP-bound spinach (pdb:8ruc) and R. palustris Rubisco (pdb:4lf1). One large subunit dimer pair (in red and cyan) is shown with helices represented by cylinders. Key segments are colored as follows: βC-βD loop, yellow; Loop 6, blue; C-terminal strand, orange. Right panels: Close-up of the active site highlighting differences in Loop 6 (in blue) closure between Form I and Form II Rubisco. Key residues and interactions are highlighted. Bound CABP is shown in ball and stick representation. The following indicated residues are conserved and functionally equivalent (Form I/Form II): E60/E49; K334/K330).

Figure 4

Current models of Rubisco activase function. (A) Bottom view of the different Rca hexameric models showing helices in cylinder view. Adjacent subunits are colored differently (B) Top view of the Rca models in surface representation. Residues known to be involved in protein-protein interactions with Rubisco are colored in magenta for red and green-type Rca. (C) Current mechanistic models for the different Rca systems. See text for details. Known Rca interacting segments on Rubisco are shown in red (RbcL C-tail) and yellow (interacting βC-βD loop residues). Red-type Rca /Rubisco, PDB:3ZUH/1BXN; Green-type Rca/Rubisco PDB:3ZW6/8RUC; CbbQ/Form IA Rubisco, PDB:5C3C/1SVD.