Structural elements of cyanobacterial co-factor-independent phosphoglycerate mutase that mediate regulation by PirC.

Abstract

The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been identified as a regulating key point in the carbon storage metabolism of cyanobacteria. Upon nitrogen starvation, the iPGAM is inhibited by the P_II-interacting regulator PirC, which is released from its interaction partner P_II due to elevated 2-oxoglutarate levels. In silico analysis of 338 different iPGAMs revealed a deep-rooted distinctive evolution of iPGAMs in cyanobacteria. Remarkably, cyanobacterial iPGAMs possess a unique loop structure and an extended C-terminus. Our mass photometry analysis suggests that iPGAM forms a complex with three individual PirC monomers. Biolayer interferometry revealed that the PirC-iPGAM complex is affected by the unique loop and the C-terminal structural elements of iPGAM. A C-terminally truncated iPGAM enzyme showed loss of control by PirC and twofold increased enzymatic activity compared to the iPGAM-WT (wild type), as demonstrated by enzyme assays. By contrast, deleting the loop structure significantly reduced the activity of this variant. Physiological experiments were carried out with different iPGAM variant strains of Synechocystis, in which these structural elements were deleted. The strain expressing the C-terminally truncated iPGAM showed a similar overproduction of polyhydroxybutyrate as deletion of the iPGAM regulator PirC. However, in contrast to the latter, these strains showed higher overall biomass accumulation, making them a better chassis for the production of polyhydroxybutyrate or other valuable substances than the PirC-deficient mutant.IMPORTANCEThe primordial cyanobacteria were responsible for developing oxygenic photosynthesis early in evolution. In the pathways of fixed carbon allocation, the co-factor-independent phosphoglycerate mutase (iPGAM) plays a crucial role by directing the first CO₂ fixation product, 3-phosphoglycerate, toward central anabolic glycolytic-derived pathways. This work reveals a distinct evolution of iPGAM within oxygenic photosynthetic organisms. We have identified two specific segments in cyanobacterial iPGAMs that affect the control of iPGAM activity through its specific interactor protein PirC. This understanding of iPGAM has allowed us to engineer cyanobacterial strains with altered carbon fluxes. Since cyanobacteria can directly convert CO₂ into valuable products, our results demonstrate a novel approach for developing a chassis for biotechnical use.