Abstract
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the best studied enzymes. It is crucial for photosynthesis, and thus for all of biosphere’s productivity. There are four isoforms of this enzyme, differing by amino acid sequence composition and quaternary structure. However, there is still a group of organisms, dinoflagellates, single-cell eukaryotes, that are confirmed to possess Rubisco, but no successful purification of the enzyme of such origin, and hence a generation of a crystal structure was reported to date. Here, we are using in silico tools to generate the possible structure of Rubisco from a dinoflagellate representative, Symbiodinium sp. We selected two templates: Rubisco from Rhodospirillum rubrum and Rhodopseudomonas palustris. Both enzymes are the so-called form II Rubiscos, but the first is exclusively a homodimer, while the second one forms homo-hexamers. Obtained models show no differences in amino acids crucial for Rubisco activity. The variation was found at two closely located inserts in the C-terminal domain, of which one extends a helix and the other forms a loop. These inserts most probably do not play a direct role in the enzyme’s activity, but may be responsible for interaction with an unknown protein partner, possibly a regulator or a chaperone. Analysis of the possible oligomerization interface indicated that Symbiodinium sp. Rubisco most likely forms a trimer of homodimers, not just a homodimer. This hypothesis was empowered by calculation of binding energies. Additionally, we found that the protein of study is significantly richer in cysteine residues, which may be the cause for its activity loss shortly after cell lysis. Furthermore, we evaluated the influence of the loop insert, identified exclusively in the Symbiodinium sp. protein, on the functionality of the recombinantly expressed R. rubrum Rubisco. All these findings shed new light onto dinoflagellate Rubisco and may help in future obtainment of a native, active enzyme.
Highlights
Ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco) is an enzyme employed by plants, algae, cyanobacteria and other autotrophic organisms to incorporate CO2 into organic compounds, it is one of the key photosynthetic enzymes
Extends over the bound RuBP trapping it underneath; the C-terminal tail of RbcL stretches across the subunit and pins down loop 6, closing the active site, which results in a closed conformation of Rubisco
Our study indicated that there is a high homology of dinoflagellate Rubisco to the same enzyme of R. palustris, shown recently to be a hexamer [30]
Summary
Ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco) is an enzyme employed by plants, algae, cyanobacteria and other autotrophic organisms to incorporate CO2 into organic compounds, it is one of the key photosynthetic enzymes. Rubisco catalyses carboxylation reaction, during which it assimilates CO2 and an oxygenation reaction, in which it oxidizes the substrate. In both reactions, the substrate is ribulose-1,5-bisphosphate (RuBP). Lysine in the active site of Rubisco must first be carboxylated by a non-substrate CO2 molecule, followed by the binding of a Mg2+ ion. This process is called carbamylation and serves to position the substrate RuBP for an efficient electrophilic attack by the second CO2 molecule that will be fixed in the Calvin-Benson cycle (CBB) cycle upon RuBP binding. Besides RuBP, Rubisco can bind other molecules like carboxyarabinitol-1,5-bisphosphate (CABP), which is a tight-binding inhibitor of this enzyme, making the active site of carbamylated or decarbamylated Rubisco adopt a closed conformation, and downregulating Rubisco’s activity [2,3,4]
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