Purification by Ni2+ Affinity Chromatography, and Functional Reconstitution of the Transporter for N-Acetylglucosamine of Escherichia coli

Abstract

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.

An Alternative Route for Recycling of N -Acetylglucosamine from Peptidoglycan Involves the N -Acetylglucosamine Phosphotransferase System in Escherichia coli

“…Mutations in nagE reduce GlcNAc(6P) levels in strains carrying nagA mutations. Another enzyme known to produce GlcNAc6P is the nagE-encoded PTS transporter for GlcNAc (19). Introduction of the nagE mutation together with the nagA mutation (mutant group 5) resulted in an approximately 60% decrease in GlcNAc(6P) levels ( Fig.…”

Phage display selection of peptides against enzyme I of the phosphoenolpyruvate–sugar phosphotransferase system (PTS)

“…Bacterial strains, M13 phage display libraries and media E. coli K-12 strains used were K91 Kan r (Kanamycin) (HfrC thi ), UT580 ⌬(lac-proAB ), supD ; f ЈlacI (Mao et al, 1995), and LR2-168⌬G (pJFEH6) (Mukhija and Erni, 1996). M13 phage display libraries expressing 6-mer, 15-mer and 10-mer inserts in pIII were obtained from J. K. Scott, G. P. Smith and B.…”

“…GlcNAc (Mukhija and Erni, 1996). After 30 min at 37ЊC, the reaction was stopped and GlcNAc-6-phosphate was separated from free GlcNAc by anion exchange chromatography on AG-1-X2 (50-100 mesh, Biorad) (Kundig and Roseman, 1971).…”

“…II GlcNAc was purified by Ni 2þ -NTA agarose (Qiagen) affinity chromatography as described in Mukhija and Erni (1996).…”

Lipid dependencies, biogenesis and cytoplasmic micellar forms of integral membrane sugar transport proteins of the bacterial phosphotransferase system

“…There is consequently a need for intracellular phosphorylation (Saier & Schmidt, 1981). In a reconstituted system, Mukhija & Erni (1996) showed that the nonvectorial sugar phosphorylation reaction exhibits about 126 lower affinity for the sugar substrate than the vectorial process, but that the maximal reaction rate for the former process is 2.56 greater than for the latter. Thus, while the cytoplasmic forms of PTS enzymes II clearly can provide the function of nonvectorial phosphorylation, it is also possible that the membrane-integrated forms can catalyse this process.…”