The Chitin Disaccharide,N,N′-Diacetylchitobiose, Is Catabolized byEscherichia coli and Is Transported/Phosphorylated by the Phosphoenolpyruvate:Glycose Phosphotransferase System

Nemat O Keyhani,Lai-Xi Wang,Y.C Lee,Saul Roseman

doi:10.1074/jbc.m001043200

Nemat O Keyhani, Lai-Xi Wang + Show 2 more

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https://doi.org/10.1074/jbc.m001043200

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Abstract

We have previously reported that wild type strains of Escherichia coli grow on the chitin disaccharide N,N'-diacetylchitobiose, (GlcNAc)(2), as the sole source of carbon (Keyhani, N. O., and Roseman, S. (1997) Proc. Natl. Acad. Sci., U. S. A. 94, 14367-14371). A nonhydrolyzable analogue of (GlcNAc)(2,) methyl beta-N, N'-[(3)H]diacetylthiochitobioside ([(3)H]Me-TCB), was used to characterize the disaccharide transport process, which was found to be mediated by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). Here and in the accompanying papers (Keyhani, N. O., Boudker, O., and Roseman, S. (2000) J. Biol. Chem. 275, 33091-33101; Keyhani, N. O., Bacia, K., and Roseman, S. (2000) J. Biol. Chem. 275, 33102-33109; Keyhani, N. O., Rodgers, M., Demeler, B., Hansen, J., and Roseman, S. (2000) J. Biol. Chem. 275, 33110-33115), we report that transport of [(3)H]Me-TCB and (GlcNAc)(2) involves a specific PTS Enzyme II complex, requires Enzyme I and HPr of the PTS, and results in the accumulation of the sugar derivative as a phosphate ester. The phosphoryl group is linked to the C-6 position of the GlcNAc residue at the nonreducing end of the disaccharide. The [(3)H]Me-TCB uptake system was induced only by (GlcNAc)(n), n = 2 or 3. The apparent K(m) of transport was 50-100 micrometer, and effective inhibitors of uptake included (GlcNAc)(n), n = 2 or 3, cellobiose, and other PTS sugars, i.e. glucose and GlcNAc. Presumably the PTS sugars inhibit by competing for PTS components. Kinetic properties of the transport system are described.

Highlights

Characterization of Phosphorylated Me-TCB and Phosphorylated (GlcNAc)2—To isolate and characterize phospho-MeTCB, the reaction mixture described above was scaled up to 5 mg of Me-TCB
There was no detectable phosphorylation of Me-TCB by toluenized cells of the mutant strain Xm1.4 under any of the conditions tested, whereas similar preparations of Xm1.4:pCBU7.3 phosphorylated Me-TCB at 2–3-fold the rate observed with the wild type cells
The product derived from phospho-Me-TCB was eluted at the same position as standard GlcNAc-6-P. These data indicate that the product of Me-TCB transport by E. coli is phospho-Me-TCB, with the phosphate linked to the C-6 position of the nonreducing terminal sugar

Summary

EXPERIMENTAL PROCEDURES

We have reported that wild type Escherichia coli and a mutant unable to transport GlcNAc can utilize the chitin disaccharide, N,NЈ-diacetylchitobiose or (GlcNAc)[21] as the sole source of carbon for growth (6).[2]. Each reaction mixture (50 ␮l) contained phosphoenolpyruvate (PEP) or ATP (10 mM), 50 mM Tris-HCl buffer, pH 8.0, 10 mM MgCl2, 10 mM KF, 0.5 mM dithiothreitol, and 1 mM of the indicated radioactive substrate (specific activity, 103 cpm/nmol) The reactions were initiated by adding 3–5 ml (10 –20 mg protein) of the cell suspension and terminated by placing tubes in a boiling water bath for 5 min. PTS assay mixtures (50 ␮l) contained 5 mM PEP (or ATP as a control), 50 mM Tris-HCl buffer, pH 8.0, 5 mM MgCl2, 10 mM KF, 0.5 mM dithiothreitol, 1 mM indicated radioactive substrate (specific activity, 103 cpm/nmol), 3–5 units (1–2 ␮g) of Enzyme I, 5–10 ␮g of HPr, and 0.5–2.5 ␮g of IIAChb. The membranes contained endogenous IIBChb, but in some experiments (e.g. Fig. 5), 5 ␮g of homogeneous IIBChb were added to increase the rate of sugar phosphorylation. Mercuric ion was removed by Dowex 50 AGX8 (Hϩ form), and the product was measured by high performance anion exchange chromatography on an system consisting of a Bio-LC (Dionex Corp., Sunnyvale, CA), and a Dionex CarboPac PA-1 column (4 ϫ 250 mm) (17)

RESULTS

Organism and substrate

Findings

DISCUSSION