Abstract 1398 A very cool and novel phosphofructokinase sheds light on the evolution of substrate specificity and on the metabolic networks in our closest prokaryotic relatives

Phosphofructokinase (PFK) is a key enzyme in the central metabolic pathway of glycolysis. PFKs are categorized according to whether they use ATP, ADP, or pyrophosphate as their phosphate donor for the sugar kinase reaction. Typically, eukaryotes use ATP-dependent PFKs while archaea have ADP-dependent PFKs. Therefore, it was fascinating when an ATP-dependent PFK was annotated in the genome of the archaeon Candidatus Prometheoarchaeum syntrophicum. Ca. P. syntrophicum is a member of the newly discovered Asgard superphylum and currently the closest cultured prokaryotic relative of eukaryotes. Thus, we hypothesised that Ca. P. syntrophicum PFK (PsyPFK) might shed light on the evolution of phosphofructokinases and phosphate donor specificity. We determined the structure of PsyPFK using X-ray crystallography. As expected, its closest structural homologues were ATP-dependent PFKs. Therefore it was a great surprise when activity assays revealed that PsyPFK had a strict preference for pyrophosphate as its phosphate donor, not ATP or ADP. The surprises kept coming when further assays revealed that PsyPFK is a multi-functional kinase/phosphatase. This single enzyme appears to perform physiological roles in glycolysis, gluconeogenesis and the pentose phosphate pathway. Indeed, its catalytic efficiency (kcat/KM) is highest for the bisphosphatase reaction that is found in gluconeogenesis, but requires an entirely different enzyme in most organisms. The phylogenetic position of Ca. P. syntrophicum suggested to us that the ATP-dependent eukaryotic PFKs may have evolved from a pyrophosphate-dependent, PsyPFK-like ancestor. We attempted to recapitulate this evolutionary trajectory using Escherichia coli, which also has ATP-dependent PFKs. We constructed an E. coli strain lacking both its PFK genes and introduced the gene for PsyPFK. As expected, this resulted in a fitness defect, which was ameliorated by serial passaging using glucose as the sole carbon source. In three separate lines, E. coli evolved back to near wild type fitness in only 100 generations. Subsequent genome sequencing revealed that PFK was bypassed altogether, by increasing flux through the pentose phosphate or Entner-Doudoroff pathways. Rather than revealing a straightforward evolutionary trajectory from pyrophosphate dependence to ATP dependence, these results showed that attempting to plug a PFK with the "wrong" phosphate donor into an existing metabolic network is difficult. In turn, this implies that phosphate donor specificity imposes a substantial barrier for horizontal transfer of PFK genes across the tree of life.