Abstract
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 EIIA gnt from Enterococcus faecalis and its unexpectedly strong complex with EIIB gnt. We analyze the activity of the complex regarding phosphoryl transfer using kinetic measurements and demonstrate by mutagenesis that His-9 of EIIA gnt is essential for this process and represents most likely the phosphoryl group carrier of EIIA gnt. With a combination of isothermal titration calorimetry (ITC), analytical ultracentrifugation (AUC), native gel electrophoresis and chemical crosslinking experiments we show that EIIA gnt and EIIB gnt form a strong 2:2 heterotetrameric complex, which seems to be destabilized upon phosphorylation of EIIB gnt.
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More From: Biochemical and Biophysical Research Communications
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