High-resolution structure of the heat-stable form-IAq RuBisCO from the thermophilic purple sulfur bacterium Thermochromatium tepidum

Abstract

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the initial carbon fixation reaction in the Calvin-Benson-Bassham cycle. Among the many forms of RuBisCOs, form-I-a protein complex containing 8 large and 8 small subunits-is the most common, representing over 90% of all known RuBisCOs. Although many form-I RuBisCO structures have been determined, no structure has been reported for a form-IAq RuBisCO. Here, we detail the structure of the heat-stable form-IAq RuBisCO from the thermophilic and anaerobic purple bacterium Thermochromatium (Tch.) tepidum at 1.55 Å resolution. The overall structure of the Tch. tepidum form-IAq RuBisCO resembles both a form-IAc RuBisCO from a chemolithotrophic sulfur bacterium and a synthetic form-I RuBisCO reconstructed from ancestral sequences. However, the Tch. tepidum enzyme shows significantly greater interactions between adjacent small subunits through their extended N-terminal domains that contain a characteristic six-residue insertion unique to form-IAq RuBisCOs. Structural differences of Tch. tepidum RuBisCO from its mesophilic relative Allochromatium vinosum, and key substitutions on the hydrophilic surface of the small subunits suggests the mechanisms of its enhanced thermostability. Our structure represents the first structure of a form-IAq RuBisCO, providing fresh clues for unraveling the evolutionary history of RuBisCO and new details for how this key enzyme remains active at elevated temperatures.

Keywords: Thermochromatium tepidum; Cryo-EM; Photosynthesis; RuBisCO.

Fig. 1

Cryo-EM structure of Tch. tepidum form-IAq RuBisCO. (a,b) The electrostatic potential density map of Tch. tepidum form-IAq RuBisCO is shown in side (a), and top (b) views. (c) Structure of an RbcL dimer of the Tch. tepidum form-IAq RuBisCO shown in cartoon mode. The active sites are marked with red circles and are enlarged in the right panel.

Fig. 2

Comparisons of amino acid sequences and structures of the RbcL subunits between Tch. tepidum form-IAq RuBisCO and the form-I RuBisCOs of Synechococcus elongatus, Cereibacter sphaeroides and Griffithsia monilis. The residue Pro²⁹⁷ and the conserved loop 6 are marked with a cyan pentagram and dashed box, respectively. Residue numbering for sequences and structural annotations (α, α-helix; β, β-strand; η, 3¹⁰-helix; TT, tight β-turns) are relative to Tch. tepidum Rubisco. Alignments were performed using ClustalWfollowed by manual curation of output files. Graphics were generated with ESPript.

Fig. 3

Inter-subunit interactions in the Tch. tepidum form-IAq RuBisCO. (a) A structural unit composed of an RbcL dimer and four associated RbcS subunits. (b) Close contacts (< 3.5 Å, hereafter) between RbcL-1 and RbcS-1. (c,d) Close contacts between RbcL-1 and RbcS-2. (e) Close contacts between RbcS-1 and RbcS-2. The conserved residues present in Tch. tepidum form-IAq RuBisCO and all three plant form-I RuBisCOs (Supplementary Fig. 7) are marked with asterisks.

Fig. 4

Sequence alignments of the RbcS subunits between Tch. tepidum form-IAq RuBisCO and other form-I RuBisCOs. Sequence similarities calculated by ClustalW are indicated on the right side of the top rows. The six-residue insertion in the N-terminal domain of form-IAq RuBisCO is boxed in red, and two additional β-strands in the form-IC and ID RuBisCOs are boxed in green. Sequence and structural annotations are the same as in Fig. 2.

Fig. 5

Comparison of the amino acid sequences of the Tch. tepidum and Alc. vinosum RuBisCO proteins. (a,b) Sequence alignment of the large subunits (a) and small subunits (b) of the Tch. tepidum and Alc. vinosum RuBisCO proteins. The substitutions distributed on the hydrophilic surface of the subunits are marked with hollow triangles (∆). Substitutions buried within the subunits are marked with filled triangles (▲). (c) Distribution of substitutions on the surfaces of the Tch. tepidum RuBisCO subunits in side and top views. Substitutions are colored in red.

Fig. 6

Structural comparisons of the two adjacent RbcS subunits (RbcS1 and RbcS2) between Tch. tepidum form-IAq RuBisCO and other representative form-I RuBisCOs. The six-residue insert in the N-terminal domain is shown in red. All other structures are superimposed with that of Tch. tepidum (gray). Form-IAc: H. neapolitanus (cyan, PDB: 7SMK), form-IB: Spinacia oleracea (green, PDB: 1RXO), form-IBc: Synechococcus elongatus PCC6301 (pink, PDB: 1RSC), form-IC: Cereibacter sphaeroides (orange, PDB: 5NV3), form-ID: Griffithsia monilis (slate blue, PDB: 8BDB). βE and βF indicate two additional β-strands in the form-IC and ID RbcS subunits. The extended βA-βB loop in the Spinacia oleracea RbcS subunit is indicated by an arrow.