DnaK and DnaJ proteins from Hsp70/40 family are involved in Rubisco biosynthesis in Synechocystis sp. PCC6803 and sustain the enzyme assembly in a heterologous system.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of carbon fixation performed by photosynthetic organisms. Form I of this enzyme found in plants and cyanobacteria is composed of eight large (RbcL) and eight small (RbcS) subunits. To form a functional enzyme, Rubisco subunits need to be properly folded, with the assistance of cellular chaperone machinery, and consecutively assembled in a strictly orchestrated manner, with the help of multiple auxiliary factors. In recent years, multiple Rubisco assembly chaperones and their function in enzyme biogenesis have been extensively characterized. Little is known about the potential specialized factors involved in Rubisco subunits folding at the pre-chaperonin stage, yet this knowledge is greatly needed for the fast and efficient testing of new Rubisco variants.Synechococcus sp. PCC 6803 Rubisco shows limited solubility and a lack of assembly in the Escherichia coli expression system. In this study, we aim to identify which additional chaperones are necessary and sufficient in sustaining the heterologous assembly of native Rubisco. Our findings prove that upon the introduction of Synechocystis DnaK2 to the E. coli system, RbcL is produced in soluble form. The addition of specific DnaJ (Sll1384) enhances this effect. We explain these combined effects based on binding constancies, measured for particular partners in vitro, as well as our analysis of the putative tertiary structure of the proteins. Our results have potential implications for Rubisco engineering.

Chaperone proteins involved in Rubisco biosynthesis / Bialka chaperonowe zaangazowane w proces biosyntezy Rubisco.

Rubisco is an enzyme found in photosynthetic organisms, which catalyse the first step of biomass accumulation: the carbon dioxide incorporation to ribulose-1,5-bisphosphate. Because of Rubisco's complicated, multimeric structure and a presence of many labile structural elements the enzyme cannot assemble to its native quaternary structure by itself. This is why the folding and assembly process of Rubisco requires the strictly organized operation of a number of auxiliary factors. Chaperone proteins take part in folding of holoenzyme subunits, subsequently they mediate in subunit oligomerisation, and in some cases chaperone proteins direct subunits to their cellular destination such as the carboxysomes or the pyrenoid. In addition to their canonical function of mediating Rubisco assembly, these chaperones are involved in additional processes such as quality control of the biosynthetic process, and regulation of organelle physiology and cellular compartments.

Insights into the Structure of Rubisco from Dinoflagellates-In Silico Studies.

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.

Is RAF1 protein from Synechocystis sp. PCC 6803 really needed in the cyanobacterial Rubisco assembly process?

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is responsible for carbon dioxide conversion during photosynthesis and, therefore, is the most important protein in biomass generation. Modifications of this biocatalyst toward improvements in its properties are hindered by the complicated and not yet fully understood assembly process required for the formation of active holoenzymes. An entire set of auxiliary factors, including chaperonin GroEL/GroES and assembly chaperones RbcX or Rubisco accumulation factor 1 (RAF1), is involved in the folding and subsequent assembly of Rubisco subunits. Recently, it has been shown that cyanobacterial RAF1 acts during the formation of the large Rubisco subunit (RbcL) dimer. However, both its physiological function and its necessity in the prokaryotic Rubisco formation process remain elusive. Here, we demonstrate that the Synechocystis sp. PCC 6803 strain with raf1 gene disruption shows the same growth rate as wild-type cells under standard conditions. Moreover, the Rubisco biosynthesis process seems to be unperturbed in mutant cells despite the absence of RbcL-RAF1 complexes. However, in the tested environmental conditions, sulfur starvation triggers the degradation of RbcL and subsequent proteolysis of other polypeptides in wild-type but not Δraf1 strains. Pull-down experiments also indicate that, apart from Rubisco, RAF1 co-purifies with phycocyanins. We postulate that RAF1 is not an obligatory factor in cyanobacterial Rubisco assembly, but rather participates in environmentally regulated Rubisco homeostasis.