Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO₂ by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO₂ molecule and binding of a Mg²⁺ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are "misfire" products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins.

Keywords: AAA+ protein; CO2 fixation; Rubisco; Rubisco activase; photosynthesis.

Figure 1

Structure and function of Rubisco. (A) Schematic depiction of photosynthesis in chloroplasts and the role of Rubisco. The light reaction and Calvin–Benson–Bassham (CBB) cycle of CO₂ fixation, as well as the side-reaction of photorespiration are shown. RuBP, ribulose-1,5-bisphosphate; 3PG, 3-phosphoglycerate; G3P, glyceraldehyde-3-phosphate; 2P-glycolate, 2-phosphoglycolate. (B) Structure of hexadecameric form I Rubisco. Side and top views of Rubisco are shown in surface representation (PDB: 1RCX, Taylor and Andersson, 1997). One antiparallel RbcL dimer with RuBP bound in the active sites is shown in ribbon representation. (C) Superposition of open and closed conformations (PDB: 1RXO and 1RCX, respectively; Taylor and Andersson, 1997) of Rubisco. In the closed state (dark green), loop 6 (cyan) covers the active site, trapping the bound RuBP (red), and is pinned down by the flexible C-terminal peptide (pink) that stretches across the RbcL subunit. In the open conformation (pale green), loop 6 (dark blue) is retracted and the C-terminal peptide (pink) is disordered.

Figure 2

Rubisco regulation by Rca. (A) Regulation of Rubisco activity and inhibition by sugar phosphates. E, the non-carbamylated enzyme; ECM, the carbamylated and Mg²⁺ ion-bound enzyme; EI, the sugar phosphate inhibited E form; ECMI, the inhibited ECM form; Rca, Rubisco activase. Figure reproduced from reference Bracher et al. (2017). (B) Phylogenetic tree of selected Rubisco RbcL sequences. The green-type enzymes encompass form IA and IB, and the red-type enzymes form IC and ID. The RbcL C-terminal sequences and their associated Rca's are indicated. X represents variable residues. Rca's from species indicated in bold have been characterized biochemically and/or structurally and are described in this review. The phylogenetic tree was calculated by multiple sequence alignment using T-Coffee (Notredame et al., 2000) and the diagram was generated by the software Dendroscope (Huson and Scornavacca, 2012). Form IA (prokaryote): M. purpuratum, Marichromatium purpuratum; H. marinus, Hydrogenovibrio marinus; T. crunogena, Thiomicrospira crunogena; H. neapolitanus, Halothiobacillus neapolitanus; N. winogradskyi, Nitrobacter winogradskyi; N. europaea, Nitrosomonas europaea; T. denitrificans, Thiobacillus denitrificans; A. ferrooxidans, Acidithiobacillus ferrooxidans; A. vinosum, Allochromatium vinosum; T. marina, Thiocapsa marina; T. mobilis, Thioflavicoccus mobilis; T. intermedia, Thiomonas intermedia. Form IB (eukaryote): Z. mays, Zea mays; T. aestivum, Triticum aestivum; O. sativa, Oryza sativa; S. oleracea, Spinacia oleracea; P. vulgaris, Phaseolus vulgaris; G. hirsutum, Gossypium hirsutum; N. tabacum, Nicotiana tabacum; B. oleracea, Brassica oleracea; A. thaliana, Arabidopsis thaliana. Form IB (prokaryote): C. reinhardtii, Chlamydomonas reinhardtii; Syn. PCC7502, Synechococcus sp. PCC 7502; F. contorta, Fortiea contorta; N. punctiforme, Nostoc punctiforme; C. stagnale, Cylindrospermum stagnale; Syn. PCC6803, Synechocystis PCC6803; Syn. PCC7002, Synechococcus PCC7002; Syn. PCC6301, Synechococcus PCC6301. Form ID (eukaryote): D. baltica, Durinskia baltica; O. sinensis, Odontella sinensis; T. oceanica, Thalassiosira oceanica; T. pseudonana, Thalassiosira pseudonana; G. partita, Galdieria partita; G. sulphuraria, Galdieria sulphuraria; P. purpurea, Porphyra purpurea; G. monilis, Griffithsia monilis; C. merolae, Cyanidioschyzon merolae. Form IC (prokaryote): X. flavus, Xanthobacter flavus; R. pickettii, Ralstonia pickettii; R. eutropha, Ralstonia eutropha; A. methanolica, Acidomonas methanolica; R. sphaeroides, Rhodobacter sphaeroides.

Figure 3

The prokaryotic Rca of red-type form IC Rubisco. (A) Schematic representation of the domain structure of Rca from R. sphaeroides. (B) Crystal structure of the monomer (PDB: 3SYL, Mueller-Cajar et al., 2011) shown in ribbon representation. The α/β and α-helical subdomains of the AAA+ core are indicated, as well as the N-terminal extension (N-ext.) of RsRca. The positions of the canonical pore loop, ADP (cyan) and the allosteric regulator, RuBP (yellow), are also indicated. (C) Top and side views of the RsRca hexameric model superposed on the electron microscopic reconstruction, with alternating subunits shown in two shades of red (EMDB EMD-1932; PDB 3ZUH, Mueller-Cajar et al., 2011). (D) Model of the putative storage form of prokaryotic Rca (Mueller-Cajar et al., 2011) from red-type form IC and its conversion to active hexamer. In the absence of photosynthetic activity (dark period), the concentration of free RuBP is low and Rca populates a helical assembly with no ATPase activity, avoiding unnecessary ATP consumption. Activation of photosynthesis results in the accumulation of free RuBP, reaching millimolar concentration (Von Caemmerer and Edmondson, 1986). Free RuBP binds to Rca, inducing its rearrangement to the catalytically competent hexamer. (E) Model of the mechanism of prokaryotic Rca from red-type form IC Rubisco. The active Rca hexamer interacts with inhibited Rubisco via its highly conserved top surface and concomitantly transiently pulls the extended C-terminal tail of the RbcL subunit into the central pore (CP). This action is mediated by the ATPase activity of Rca and results in the destabilization of the Rubisco active site, releasing the inhibitory sugar phosphate. Rca is displayed as in (C). Rubisco (PDB: 4F0K, Stec, 2012) is shown in surface representation with the RbcL and RbcS subunits in different shades of pink. The RbcL C-termini are drawn as lines in red.

Figure 4

The prokaryotic Rca of the green-type form IA Rubisco. (A) Schematic representation of the domain structure of Rca from H. neapolitanus and its adapter protein (CbbO). (B) Crystal structure of the monomer (PDB: 5C3C, Sutter et al., 2015) shown in ribbon representation. The α/β and α-helical subdomains of the AAA+ core are indicated, as well as the positions of the pore loops and ADP (cyan). (C) Top and side views of the HnRca hexameric model (PDB: 5C3C, Sutter et al., 2015) superposed on the electron microscopic reconstruction of the Rca hexamer from A. ferrooxidans (EMDB: EMD-6477, Tsai et al., 2015). Alternating subunits shown in two shades of blue. (D) Model of the mechanism of prokaryotic Rca from green-type form IA Rubisco. The Rca hexamer interacts with inhibited Rubisco via the VWA domain of its adapter protein CbbO, recognizing the exposed acidic residue Asp82 (marine blue) on the RbcL subunit of Rubisco. Whether the central pore (CP) then engages the C-terminal tail of the RbcL subunit, remains unclear. The hexameric HnRca is displayed as in (C). Rubisco (PDB: 1SVD, Kerfeld et al., 2004) is shown in surface representation with the RbcL and RbcS subunits in different shades of blue. The RbcL C-termini are represented by blue lines.

Figure 5

The eukaryotic Rca of the green-type form IB Rubisco. (A) Schematic representation of the domain structure of Rca from N. tabacum. (B) Crystal structure of the monomer (PDB: 3T15, Stotz et al., 2011) shown in ribbon representation. The α/β and α-helical subdomains of the AAA+ core are indicated, as well as the positions of the pore loops and the specificity helix H9. The positions of the N-terminal domain (N-domain) and the flexible C-terminal extension (C-Ext.), not present in the crystallized construct, are also indicated. (C) Top and side views of the NtRca hexameric model (PDB: 3ZW6, Stotz et al., 2011) superposed on the electron microscopic reconstruction (EMDB: EMD-1940, Stotz et al., 2011). The unfilled electron density at the top of the hexamer probably represents the N-domains. Alternating subunits are shown in two shades of green and the specificity helix (H9) in purple. (D) Model of the mechanism of eukaryotic Rca from green-type form IB Rubisco. The Rca hexamer interacts with inhibited Rubisco via the N-domain and H9 recognizes the exposed basic residue Arg89 (dark green) on the RbcL subunit. Whether the central pore (CP) engages the C-terminal tail of RbcL, remains unclear. The hexameric NtRca is displayed as in (C). Rubisco (PDB: 1EJ7, Duff et al., 2000) is shown in surface representation with the RbcL and RbcS subunits in different shades of green. The RbcL C-termini are shown as green lines.