Purification by Ni2+ affinity chromatography, and functional reconstitution of the transporter for N-acetylglucosamine of Escherichia coli.
The N-acetyl-D-glucosamine transporter (IIGlcNAc) of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported GlcNAc. IIGlcNAc of Escherichia coli containing a carboxyl-terminal affinity tag of six histidines was purified by Ni2+ chelate affinity chromatography. 4 mg of purified protein was obtained from 10 g (wet weight) of cells. Purified IIGlcNAc was reconstituted into phospholipid vesicles by detergent dialysis and freeze/thaw sonication. IIGlcNAc was oriented randomly in the vesicles as inferred from protein phosphorylation studies. Import and subsequent phosphorylation of GlcNAc were measured with proteoliposomes preloaded with enzyme I, histidine-containing phosphocarrier protein, and phosphoenolpyruvate. Uptake and phosphorylation occurred in a 1:1 ratio. Active extrusion of GlcNAc entrapped in vesicles was also measured by the addition of enzyme I, histidine-containing phosphocarrier protein, and phosphoenolpyruvate to the outside of the vesicles. The Km for vectorial phosphorylation and non-vectorial phosphorylation were 66. 6 +/- 8.2 microM and 750 +/- 19.6 microM, respectively. Non-vectorial phosphorylation was faster than vectorial phosphorylation with kcat 15.8 +/- 0.9 s-1 and 6.2 +/- 0.7 s-1, respectively. Using exactly the same conditions, the purified transporters for mannose (IIABMan, IICMan, IIDMan) and glucose (IICBGlc, IIAGlc) were also reconstituted for comparison. Although the vectorial transport activities of IICBAGlcNAc and IICBGlc. IIAGlc are inhibited by non-vectorial phosphorylation, no such effect was observed with the IIABMan.IICMan.IIDMan complex. This suggests that the molecular mechanisms underlying solute transport and phosphorylation are different for different transporters of the phosphotransferase system.
Mukhija
S
S
Institute of Biochemistry, University of Bern, CH-3012 Bern, Switzerland.
Erni
B
B
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D008081:Liposomes; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D009532:Nickel; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C081515:acetylglucosamine transporter