Gain-of-function mutations in 1-phosphofructokinase (FruK) re-establish glycolysis in bacteria

Secondary (promiscuous) activities provide enzymes with a reservoir of latent catalytic functions, which can be leveraged during selective pressure. Here we retrace the re-emergence of glycolysis from the promiscuous activity of 1-phosphofructokinase (FruK). We evolved a hexose-degradation-deficient Escherichia coli strain on glucose and recovered non-synonymous mutations in fruK. Reverse engineering, gene deletions and transcript quantification identified a modified and strongly overexpressed fruK as the determinant of the growth phenotype. Purified FruK variants phosphorylated fructose-6-phosphate in vitro, the best variant with ~170-fold improved catalytic efficiency over the wildtype enzyme. Crystal structures at up to 1.4 angstrom resolution captured FruK with ADP and either F1P, F6P or FBP bound. They identify a single active-site lysine that excludes the alternative substrate, and which E. coli FruK retains despite forgoing a 26 % gain in efficiency on its own native reaction. Since F1P, the product of the reverse reaction of FruK, is the effector of the global regulator Cra, we propose that this residue keeps glycolytic flux separate from transcriptional control. The same active-site motifs occur naturally across FruK clades, and a hexose-degradation-deficient Corynebacterium glutamicum strain converged on the same adaptation, indicating a common strategy across the Gram divide. The committed step of EMP glycolysis therefore lies a single substitution away within a widespread sugar kinase family, where it appears to be actively suppressed.