GK PUA Domain Modulates Cables, Cooperativity, and Proline Feedback

In E. coli , the committed step of proline biosynthesis is initiated by γ‐glutamate kinase (GK); however, the regulatory logic governing this enzyme remains poorly understood. E. coli GK (EcGK) possesses an enigmatic C‐terminal PUA domain—a motif historically linked to RNA binding but uncharacterized in this context. Here, we demonstrate that EcGK forms metabolic filaments and further assembles into four‐stranded metabolic cables. Cryo‐EM structures of EcGK filaments reveal two assembly interfaces: one analogous to the eukaryotic P5CS “hook” motif and a novel interface mediated by the PUA domain. Filament formation hinders substrate binding, whereas higher‐order cable assembly introduces additional cooperativity in substrate binding. Moreover, we resolve the first proline‐bound EcGK structure, revealing the function of the PUA domain and the E135‐D148 loop in proline feedback inhibition. Thus, the PUA domain plays dual roles in EcGK regulation: it drives filament/cable assembly to reshape substrate binding kinetics, and also facilitates the E135–D148 loop to enable high sensitivity to proline feedback inhibition. In E. coli , the PUA domain thus modulates proline biosynthesis within physiological ranges of substrates and products.