Metabolic regulation and structural mechanism of glutamine synthetase AMPylation
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In bacteria, glutamine synthetase (GS) is the main ammonium assimilation enzyme. Its activity is tightly regulated according to cellular energy status and carbon–nitrogen balance through reversible AMPylation catalyzed by the bifunctional enzyme GlnE, which is controlled by the signal transducer GlnB protein. Although GS AMPylation has been extensively studied, the GlnB:GlnE:GS regulatory pathway exhibits substantial plasticity among bacterial groups, and the structural basis of GS inhibition by AMPylation remains unclear. Here, we describe how carbon, nitrogen, and energy signals regulate GS AMPylation in Herbaspirillum seropedicae and uncover the structural mechanism underlying enzyme inhibition. Our data reveal that GS AMPylation is independent of unmodified GlnB, whereas uridylylated GlnB (GlnB-UMP) inhibits AMPylation under nitrogen-limiting conditions through a GlnB-GlnE complex modulated by 2-oxoglutarate. We further show that GlnE directly senses glutamine under nitrogen-sufficient conditions, with signal integration depending primarily on energy availability. To elucidate the mechanism of AMPylation GS inhibition, we solved Cryo-EM structures of unmodified and AMPylated GS in complex with MgATP and MnADP. Structural comparisons revealed that AMPylation increases the flexibility of the AMP-loop, disrupting a hydrogen-bond network that stabilizes Arg342 in the orientation required to position the ATP γ-phosphate in its catalytic conformation for efficient phosphoryl transfer to glutamate. These findings reveal how metabolic signals are integrated to regulate GS AMPylation and provide the first structural insights into the mechanism underlying bacterial GS inhibition by AMPylation.
Importance
Glutamine synthetase is the central enzyme of bacterial nitrogen assimilation and has been studied for more than six decades. Although its regulation by reversible AMPylation has long been recognized, the structure of the AMPylated enzyme has remained unknown, leaving the molecular basis of inhibition unresolved. We present the first structures of AMPylated glutamine synthetase, revealing that the modification increases the flexibility of a surface loop that disrupts the positioning of a catalytic residue required for ATP utilization. Together with the identification of an alternative signaling mechanism controlling AMPylation in Herbaspirillum seropedicae , these findings resolve a longstanding question in bacterial nitrogen metabolism and uncover unexpected diversity in its regulation.
