A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts as a small subunit mimic

Abstract

The catalytic inefficiencies of the CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L₂)₅, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L₈S₈ enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L₈S₈ enzymes between LSus of adjacent L₂ dimers, where negatively charged residues coordinate around a Mg²⁺ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L₂ dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C₃ carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB.

Keywords: X-ray crystallography; archaea; carbon fixation; metal ion-protein interaction; oligomerization; protein evolution; ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco); structure-function.

Figure 1.

MbR has its own inbuilt SSu that concentrates LSu dimers. A, comparison of the position of the Rubisco SSu and MbR RAD in relation to Rubisco LSus: top and side views of the 3D crystal structures of Spinacia oleracea (spinach) form I L₈S₈ Rubisco (LSus and SSus green and yellow, respectively; PDB code 8RUC); T. kodakarensis form II L₁₀ Rubisco (LSus, pink; PDB code 1GEH); MbR L₁₀ Rubisco (LSus, blue; RAD, red). In the top MbR structure, five L₂ dimers are arranged around a non-crystallographic 5-fold axis perpendicular to the page. B, structure of the MbR RAD rainbow-colored from blue at the C terminus to red at the N terminus. The RAD was defined from structural alignments as residues 361–389. C, potential locking mechanism between αJ of the RAD (red) and α1 of a neighboring Rubisco LSu (blue) involves the coordination of four negatively charged side chains and a solvent molecule around a magnesium ion. D, RAD (red) packs against its own LSu (dark blue) and interacts with a neighboring LSu (light blue). The interface is formed between loop residues in the RAD and residues in α1 and α2 of the neighboring LSu. Lock site, ionic, and hydrogen-bonding interactions at the interface are shown as green, yellow, and black dashed lines, respectively. The top and side views correspond to the views shown in A.

Figure 2.

Comparison of the electrostatic surface potential of L₂ Rubisco dimers from T. kodakarensis and M. burtonii. Electrostatic surface potential at the interface between L₂ dimers and at the surface that lies within the Rubisco solvent channel in MbR (A) and T. kodakarensis (B). Electrostatic surfaces are colored blue in positive regions and red in negative regions. The regions corresponding to the MbR dimer-dimer lock site and the complementary charges at the T. kodakarensis Rubisco dimer-dimer interface are indicated by solid circles. The location of the RAD is indicated by dashed circles in A.

Figure 3.

Oligomerization potential of MbR harboring site-specific mutations. Non-denaturing PAGE analyses of IMAC-purified single-MbR mutants (A) and double-MbR mutants (B) incubated with a 10× molar concentration of 2-CABP (relative to the number of Rubisco active sites). C, 2-CABP-bound wild-type MbR and putative lock site single mutants from A were incubated with increasing concentrations of EDTA. 5 μg of protein was loaded per lane. Lane m, protein molecular mass marker, sizes shown in kDa; lane C, pHUE empty-vector negative control; lane WT, wild-type MbR (positive control); lane WT-tag cleaved, wild-type MbR without a H₆-Ub tag. Protein bands corresponding to distinct MbR oligomeric states are indicated.

Figure 4.

Ligand binding to MbR. A, ligands used in this study. B, non-denaturing PAGE protein separation. Purified and activated MbR were incubated with 1 or 10× molar concentrations of 2-CABP, 4-CABP, and XuBP and 1, 10, and 1000× molar concentrations of 3-PGA. 14 μg of protein was loaded per lane. Lane m, protein molecular mass marker, sizes shown (in kDa); lane C, purified MbR incubated with crystallization buffer.

Figure 5.

Relative locations of the active and lock sites in MbR. A, surface representations of L₁₀ MbR. LSus are colored dark/light blue, and the assembly domain is highlighted in red. Left and center, side view of MbR showing lock sites at the interface between MbR dimers. Right, top view showing five lock sites. Lock and active sites are indicated by white and yellow arrowheads, respectively. B, close-up view of the green boxed region in A, right, showing the relative location of the lock sites (sticks) to the active site. Mg²⁺ ions are shown as green spheres, and 2-CABP bound at the active site is shown as ball and sticks. The helices αJ and α1 (ribbon representation) are linked to active site residues Lys-330, Ser-399, Lys-167, and Lys-193 (sticks) through defined structural elements (schematic representation). Distances between the active- and lock- site magnesium ions are indicated.

Figure 6.

Unrooted minimum evolution phylogenetic tree of Rubisco LSu sequences. The optimal unrooted Rubisco LSu tree with the sum of branch length = 12.58 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances are in the units of the number of amino acid differences per site. The analysis included 15 representative sequences from each of the forms IA–ID, form II, form III, and form IV (RLPs) Rubisco groups and subgroups and all available Rubisco sequences from the Methanosarcinales order. The sequences used for phylogenetic reconstruction, and their homology to MbR, are included in supplemental Table S2. Bootstrap values ≥95% (** = 100%, * = 95–99%) obtained after 2000 bootstrap iterations are plotted at branch points. A black arrow indicates the location of the MbR sequence.