Activity of the Enterococcus faecalis EIIAgnt PTS component and its strong interaction with EIIBgnt

Eubacteria can import and simultaneously phosphorylate a range of different carbohydrates by means of sugar specific phosphoenolpyruvate (PEP) dependent sugar phosphotransferase systems (PTSs). Here, we report the biochemical characterization of the gluconate specific PTS component EIIAgnt from Enterococcus faecalis and its unexpectedly strong complex with EIIBgnt. We analyze the activity of the complex regarding phosphoryl transfer using kinetic measurements and demonstrate by mutagenesis that His-9 of EIIAgnt is essential for this process and represents most likely the phosphoryl group carrier of EIIAgnt. With a combination of isothermal titration calorimetry (ITC), analytical ultracentrifugation (AUC), native gel electrophoresis and chemical crosslinking experiments we show that EIIAgnt and EIIBgnt form a strong 2:2 heterotetrameric complex, which seems to be destabilized upon phosphorylation of EIIBgnt.