Structural Basis for Enzyme I Inhibition by α-Ketoglutarate

Creating new bacterial strains in which carbon and nitrogen metabolism are uncoupled is potentially very useful for optimizing yields of microbial produced chemicals from renewable carbon sources. However, the mechanisms that balance carbon and nitrogen consumption in bacteria are poorly understood. Recently, alpha-ketoglutarate (alpha KG), the carbon substrate for ammonia assimilation, has been observed to inhibit Escherichia colt enzyme I (El), the first component of the bacterial phosphotransferase system (PTS), thereby providing a direct biochemical link between central carbon and nitrogen metabolism. Here we investigate the El-alpha KG interaction by NMR and enzymatic assays. We show that alpha KG binds with a K-D of similar to 2.2 M at the active site of El, acting as a competitive inhibitor. In addition, we use molecular docking simulations to derive a structural model of the enzyme inhibitor complex that is fully consistent with NMR and analytical ultracentrifugation data. We expect that the EI-alpha KG structure presented here will provide a starting point for structure-based design of El mutants resistant to alpha KG.