Carmen Peraglie (Essen / DE), Lu Shen (Essen / DE), David Podlesainski (Essen / DE), Christina Stracke (Essen / DE), Ravi Shankar Ojha (Essen / DE), Frauke Caliebe (Kassel / DE), Markus Kaiser (Essen / DE), Karl Forchhammer (Tübingen / DE), Martin Hagemann (Rostock / DE), Kirstin Gutekunst (Kassel / DE), Jacky L. Snoep (Stellenbosch / DE), Christopher Bräsen (Essen / DE), Bettina Siebers (Essen / DE)
In photosynthetic organisms, efficient carbohydrate metabolism is crucial for adapting to varying environmental conditions, particularly during transitions between light and dark cycles. Phosphofructokinase (PFK) plays a pivotal role in glycolysis, acting as one of two key regulatory points that govern the flow of metabolites via the Embden-Meyerhof-Parnas (EMP) pathway. Three different types of PFKs have been described that vary in their phosphate donor, i.e. ATP, ADP or PPi and regulatory properties [1]. Synechocystis PFK activity has been analysed in cell extracts [2], and it was classified as member of the PFK-B family, even though sequence comparisons indicated them to be members of the PFK-A family [3].
We aimed to characterize the enzymatic properties, regulatory mechanisms, and evolutionary adaptations of the two PFK isoenzymes in Synechocystis, particularly their roles in adjusting to changing growth conditions and metabolic states.
The genes encoding PFK-A1 and PFK-A2 from Synechocystis were cloned, expressed, and purified. Kinetic parameters were determined under varying substrate and effector concentrations, and the phylogenetic relationships were examined through sequence comparisons and structural modeling.
Both PFK-A1 and PFK-A2 showed exclusive dependence on ADP for catalytic activity, with distinct regulatory profiles: PFK-A1 is inhibited by 3-phosphoglycerate, while PFK-A2 is inhibited by ATP. Both of these inhibitors accumulate during photosynthesis, facilitating enzyme inactivation during light. Phylogenetic analysis identified these ADP-dependent PFK-A isoenzymes as monophyletic group within the PFK-A family, comprising cyanobacterial evolutionary lineage and structural adaptation among cyanobacteria and homologs [4].
The study provides new insights into the regulatory properties and evolution of PFK-A isoenzymes in cyanobacteria, emphasizing an adaptive mechanism for balancing glycolysis and gluconeogenesis in response to light and energy charge of the cell. In addition, their ADP dependence offers interesting potential for optimization of carbohydrate metabolism in metabolic engineering and synthetic biology approaches.
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