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

Gain-of-function mutations in 1-phosphofructokinase (FruK) re-establish glycolysis in a hexose-degradation-deficient Escherichia coli strain, yielding a ~170-fold catalytic efficiency increase.

• Why it matters: Promiscuous enzyme activities can be recruited under selection, but how latent functions re-emerge to restore a core pathway like EMP glycolysis is unclear.
• What they did: The authors evolved the Escherichia coli strain on glucose, reverse-engineered fruK variants, used gene deletions and transcript quantification, and measured in vitro phosphorylation of fructose-6-phosphate. Crystal structures at up to 1.4 angstrom with ADP plus F1P, F6P, or FBP mapped substrate binding and an active-site lysine.
• The result: The best FruK variant phosphorylated fructose-6-phosphate with ~170-fold improved efficiency, and structural data suggest the retained lysine separates glycolytic flux from Cra-linked transcriptional control. Similar active-site motifs and a convergent adaptation in a hexose-degradation-deficient Corynebacterium glutamicum strain indicate a shared strategy across Gram lineages.

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