Phosphoenolpyruvate carboxylase from the cyanobacterium Synechocystis sp. PCC 6803 is under global metabolic control by P_II signaling

Abstract

Phosphoenolpyruvate carboxylase (PEPC) is the second major carbon‐fixing enzyme in photoautotrophic organisms. PEPC is required for the synthesis of amino acids of the glutamate and aspartate family by replenishing the TCA cycle. Furthermore, in cyanobacteria, PEPC, together with malate dehydrogenase and malic enzyme, forms a metabolic shunt for the synthesis of pyruvate from PEP. During this process, CO2 is first fixed and later released again. Due to its central metabolic position, it is crucial to fully understand the regulation of PEPC. Here, we identify PEPC from the cyanobacterium Synechocystis sp. PCC 6803 (PEPC) as a novel interaction partner for the global signal transduction protein PII. In addition to an extensive characterization of PEPC, we demonstrate specific PII–PEPC complex formation and its enzymatic consequences. PEPC activity is tuned by the metabolite‐sensing properties of PII: Whereas in the absence of PII, PEPC is subjected to ATP inhibition, it is activated beyond its basal activity in the presence of PII. Furthermore, PII–PEPC complex formation is inhibited by ADP and PEPC activation by PII‐ATP is mitigated in the presence of 2‐OG, linking PEPC regulation to the cell's global carbon/nitrogen status. Finally, physiological relevance of the in vitro measurements was proven by metabolomic analyses of Synechocystis wild‐type and PII‐deficient cells.

The Novel PII-Interacting Regulator PirC (Sll0944) Identifies 3-Phosphoglycerate Mutase (PGAM) as Central Control Point of Carbon Storage Metabolism in Cyanobacteria

“…A variety of key metabolic enzymes, transcription factors and transport proteins use this signalling path to tune their activity in response to the metabolic state. In many cases, the different PII conformations can directly interact with target proteins such as the N-Acetyl-L-glutamate kinase, catalysing the committed step in arginine biosynthesis (7,8) , the acetyl-coA carboxylase (ACCase), catalysing the rate-limiting step in fatty acid biosynthesis (9), the phosphoenolpyruvate carboxylase (PEPC), which catalyses an anaplerotic CO2-fixation (10), or the glutamine-dependent NAD + synthetase (NadE) (11). In addition to the regulation of enzyme activities via PII, the trimeric protein can also influence transport activities.…”

Reconstitution of oxaloacetate metabolism in the tricarboxylic acid cycle in Synechocystis sp. PCC 6803: discovery of important factors that directly affect the conversion of oxaloacetate

“…A decrease in the concentration of MgCl 2 also led to a decrease in the yield of l ‐malate + citrate (Figure 3). Mg 2+ also acts as a cofactor for Sy PEPC, and the activity of Sy PEPC increases depending on the concentration of Mg 2 + (the substrate concentration at 50% V max , K m = 4.27 m m ) (Scholl et al ., 2020). From the above, it is thought that Mg 2+ comprehensively regulates oxaloacetate metabolism in the TCA cycle in Synechocystis 6803, and a high concentration of Mg 2+ improves the flux of citrate generation (Figure 7).…”

The novel PII-interacting protein PirA regulates flux into the cyanobacterial ornithine-ammonia cycle

“…His 8 -tagged variants of P II were prepared as previously described (79). For the preparation of recombinant PirA protein for BLI analysis, the pirA gene was cloned into XhoI and EcoRI sites of pGEX-4T-3 vector (GE Healthcare Life Sciences, Freiburg, Germany), encoding recombinant PirA with N-terminal-fused GST-tag.…”