Activity of the Enterococcus faecalis EIIA(gnt) PTS component and its strong interaction with EIIB(gnt).
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).
Brockmeier
Achim
A
AG Physiology of Microorganisms, Ruhr-Universität Bochum, 44780 Bochum, Germany.
Skopnik
Manuel
M
Koch
Brigitte
B
Herrmann
Christian
C
Hengstenberg
Wolfgang
W
Welti
Stefan
S
Scheffzek
Klaus
K
D002151:Calorimetry; D002384:Catalysis; D013293:Enterococcus faecalis / Q000201:enzymology*; D005942:Gluconates / Q000737:chemistry / Q000378:metabolism*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D014461:Ultracentrifugation
D005942:Gluconates; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030691:gluconic acid