Structure of the photosynthetic Calvin-Benson-Bassham sedoheptulose-1,7-bisphosphatase SBPase from the model microalga Chlamydomonas reinhardtii

Reviewer #1 (Public Review):

In this study, Le Moigne and coworkers shed light on the structural details of the Sedoheptulose-1,7-Bisphosphatase (SBPase) from the green algae Chlamydomonas reinhardtii. The SBPase is part of the Calvin cycle and catalyzes the dephosphorylation of sedoheptulose-1,7-bisphosphate (SBP), which is a crucial step in the regeneration of ribulose-1,5-bisphosphate (RuBP), the substrate for Rubisco. The authors determine the crystal structure of the CrSBPase in an oxidized state. Based on this structure, potential active site residues and sites of post-translational modifications are identified. Furthermore, the authors determine the CrSBPase structure in a reduced state revealing the disruption of a disulfide bond in close proximity to the dimer interface. The authors then use molecular dynamics (MD) to gain insights into the redox-controlled dynamics of the CrSBPase and investigate the oligomerization of the protein using small-angle X-ray scattering (SAXS) and size-exclusion chromatography. Despite the difference in oligomerization, disruption of this disulfide bond did not impact the activity of CrSBPase, suggesting additional thiol-dependent regulatory mechanisms modulating the activity of the CrSBPase.

The authors provide interesting new findings on a redox-mechanism that modulates the oligomeric behavior of the SBPase, however without investigating this potential mechanism in more detail. The conclusions of this manuscript are mostly supported by the data, but they should be more carefully evaluated in respect to what is known from other systems as e.g. the moss Physcomitrella patens. This is especially of interest, as SBPase was previously reported to be dimeric, whereas for FBPase a dimer/tetramer equilibrium has been observed.

1.) Given that PpSBPase has been already characterized in detail, the authors should provide a more rigorous comparison to the existing data on SBPases. This includes a more conclusive structural comparison but also the enzymatic assays should be compared to the findings from P. patens. Do the authors observe differences between the moss and the chlorophyte systems, maybe even in regard to the oligomerization of the SBPase?

2.) The authors should include the control experiments (untreated SBPase) and the assays performed with mutant versions of the SBPase, which are currently only mentioned in the text or not shown at all.

3.) The representation of the structure in figures (especially Figures 1 and 3) should be adjusted to match the author's statements. In Figure 1, the angle from which the structure is displayed changes over the entire figure making it difficult to follow especially as a non-structural biologist. Furthermore, important aspects of the structure mentioned in the text are not labeled and should be highlighted, by e.g. a close-up. Same holds true for Figure 3 that currently mostly shows redundant information.

4.) The authors state that mutation of C115 and C120 to serine destabilize the dimer formation, while more tetramer and monomer is formed. As the tetramer is essentially a dimer of dimers, the authors should elaborate how this might work mechanistically. In my opinion, dimer formation is a prerequisite for tetramer formation and the two mutations rather stabilize the tetramer instead of destabilizing the dimer.

Reviewer #2 (Public Review):

The central theme of the manuscript is to report on the structure of SBPase - an enzyme central to the photosynthetic Calvin-Benson-Bassham cycle. The authors claim that the structure is first of its kind from a chlorophyte Chlamydomonas reinhardtii, a model unicellular green microalga. The authors use a number of methods like protein expression, purification, enzymatic assays, SAXS, molecular dynamics simulations and xray crystallography to resolve a 3.09 A crystal structure of the oxidized and partially reduced state. The results are supported by the claims made in the manuscript. One of the main weakness of the work is the lack of wider discussion presented in the manuscript. While the structure is the first from a chlorophyte, it is not unique. Several structures of SBPase are available. As the manuscript currently reads, the wider context of SBPase structures available and comparisons between them is missing from the manuscript. Another important point is that the reported structure of crSBPase is 0.453A away from the alphafold model. Though fleetingly mentioned in the methods section, it should be discussed to place it in the wider context.