Investigating the regulation of the carbon switch in central metabolism of Synechocystis sp. PCC 6803

Dependent on the light/nutrient conditions Synechocystis is able to grow auto-, hetero-, and mixotrophically for which the reversibility of sugar metabolism particularly the Embden-Meyerhof-Parnas pathway (EMP) is essential. To switch between anabolic and catabolic direction sophisticated regulation is required and also the interplay of e.g. EMP and Calvin cycle for CO₂ fixation must be tightly balanced. The antagonistic PFK and FBPase operate exclusively in the catabolic or anabolic direction of the EMP, respectively, and PFK is the classical control point of glycolysis. Two paralogous copies of both enzymes are present in Synechocystis. Furthermore, the reversible phosphoglucose isomerase (PGI) has been discussed to direct fluxes between EMP and CBB under different growth conditions. However, the control points and the function and regulatory capacities of the enzymes are not established in Synechocystis.

Herein, the regulatory functions of the PFK and FBPase isoenzymes as well as the PGI were analysed. Pfk-A1, Pfk-A2, Fbpase1 and Pgi from Synechocystis were overproduced in E. coli, purified and characterized with respect to substrate specificity, kinetic and regulatory properties.

Both PFKs are ADP dependent, thereby defining a new class of ADP-dependent PFK-A superfamily. PFK-A1 is inhibited by 3-phosphoglycerate (3PG), and PFK2 by ATP. FBPase1 is not regulated by various effectors. The PGI displays a preference for the gluconeogenic direction and is inhibited by erythrose 4-phosphate (E4P).

Our investigations reveal that both PFKs are active under conditions of low energy-charge, facilitating the breakdown of glycogen/D-glucose. The inhibition of PFK-A1 and PFK-A2 by 3PG and ATP, respectively, enables rapid generation of CO₂ acceptor molecule. The sustained FBPase1 activity in presence of various effectors suggests its housekeeping role to enable reversibility of the EMP pathway in response to substrate availability/growth conditions. E4P enables PGI function as a valve to balance the non-oxidative pentose phosphate pathway intermediates and glycogen synthesis.

Feng et al., FEBS J (2014) 281.3: 916-926Schulze et al., Microb Cell Fact (2022); 21:699