NahK/GlmU fusion enzyme: characterization and one-step enzymatic synthesis of UDP-N-acetylglucosamine.

The availability of uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a prerequisite for the GlcNAc-transferase-catalyzed glycosylation reaction. UDP-GlcNAc has already been synthesized using an N-acetylhexosamine 1-kinase (NahK) and a GlcNAc-1-P uridyltransferase (truncated GlmU) and here, a fusion enzyme was constructed with truncated GlmU and NahK. After determination of the optimum catalytic condition (pH 8.0 at 40 °C), the fusion enzyme was used to synthesize UDP-GlcNAc in a single step with a yield of 88 % from GlcNAc, ATP and UTP. Furthermore, a simplified purification method was demonstrated using separation by gel filtration after by-product digestion with alkaline phosphatase. An overall yield of 77 % and a purity of over 90 % were achieved.

Synthesis of FUDP-N-acetylglucosamine and FUDP-glucose in Saccharomyces cerevisiae cells treated with 5-fluorouracil.

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.

Hsp70-GlcNAc-binding activity is released by stress, proteasome inhibition, and protein misfolding.

Numerous recent works strengthen the idea that the nuclear and cytosolic-specific O-GlcNAc glycosylation protects cells against injuries. We have first investigated O-GlcNAc level and Hsp70-GlcNAc-binding activity (HGBA) behaviour after exposure of HeLa and HepG(2) cells to a wide variety of stresses. O-GlcNAc and HGBA responses were different according to the stress and according to the cell. HGBA was released for almost all stresses, while O-GlcNAc level was modified either upwards or downwards, depending to the stress. Against all expectations, we demonstrated that energy charge did not significantly vary with stress whereas UDP-GlcNAc pools were more dramatically affected even if differences in UDP-GlcNAc contents were not correlated with O-GlcNAc variations suggesting that O-GlcNAc transferase is itself finely regulated during cell injury. Finally, HGBA could be triggered by proteasome inhibition and by L-azetidine-2-carboxylic acid (a proline analogue) incorporation demonstrating that protein misfolding is one of the key-activator of this Hsp70 property.

One-day enzymatic synthesis and purification of UDP-N- [1-14C]acetyl-glucosamine.

UDP-GlcN[1-14C]Ac was synthesized in a single enzymatic reaction from [1-14C]acetate and commercially available precursors on both a microcurie (micromole) and a millicurie (millimole) scale. The reaction was catalyzed by the action of acetyl coenzyme A synthetase, inorganic pyrophosphatase, and the bifunctional Escherichia coli GlmU protein. Within 2 h 86 to 94% reaction is attained, and it approaches 99% completion overnight. GlmU protein was prepared in the form of a fusion suitable for nickel chelate affinity chromatography. Several methods were developed for rapid purification of UDP-GlcN[1-14C]Ac: an HPLC method handled micromole (microcurie) loads. Alternatively, ion exchange chromatography over DOWEX AG1 X-2 using a batch elution procedure was compatible with millimole (millicurie) amounts of radiolabel and yielded both chemically and radiochemically homogeneous UDP-GlcN[1-14C]Ac. These methods allow laboratories to quickly produce and purify microcurie to millicurie quantities of N-acetyl-labeled UDP-GlcNAc by a choice of methods from relatively inexpensive precursors.

Characterization of UDP amino sugars as major phosphocompounds in the hyperthermophilic archaeon Pyrococcus furiosus.

The archaeon Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally at 100 degrees C by the fermentation of carbohydrates. It is known to contain high concentrations of novel intracellular solutes such as beta-mannosylglycerate and di-myo-inositol 1,1'-phosphate (DIP) (L. O. Martins and H. Santos, Appl. Environ. Microbiol. 61:3299-3303, 1995). Here, 31P nuclear magnetic resonance (NMR) spectroscopy was used to show that this organism also accumulates another type of phospho compound, as revealed by a major multiplet signal in the pyrophosphate region. The compounds were purified from cell extracts of P. furiosus by anion-exchange and gel filtration chromatographic procedures and were structurally analyzed by 1H, 13C, and 31P NMR spectroscopy. They were identified as two uridylated amino sugars, UDP N-acetylglucosamine and UDP N-acetylgalactosamine. Unambiguous characterizations and complete assignments of 1H and 13C resonances from such sugars have not been previously reported. In vitro 31P NMR spectroscopic analyses showed that, in contrast to DIP, which is maintained at a constant intracellular concentration (approximately 32 mM) throughout the growth phase of P. furiosus, the UDP amino sugars accumulated (to approximately 14 mM) only during the late log phase. The possible biochemical roles of these compounds in P. furiosus are discussed.

Stimulation of the methylcoenzyme M reduction by uridine-5'-diphospho-sugars in cell-free extracts of Methanobacterium thermoautotrophicum (strain delta H).

The hydrogen-dependent reduction of methylcoenzyme M catalyzed by coenzyme-depleted cell-free extracts of Methanobacterium thermoautotrophicum was stimulated by micromolar concentrations of a UDP-disaccharide present in the organism. The compound was isolated and identified as UDP-1-O-alpha-D-2-acetamido-2-deoxyglucopyranose (UDPGlcpNAc) glycosidically linked to 2-acetamido-2-deoxymannopyranosyluronic acid. Maximal stimulation was observed when both the UDP-disaccharide and mercaptoheptanoylthreonine phosphate were present in the reaction mixtures. The UDP derivative isolated was not specific in its action: other UDP-sugars tested in micromolar concentrations stimulated the methylcoenzyme M reduction to the same extent. The activated sugars presumably substitute for ATP, which is usually required in much higher concentrations to activate the methylcoenzyme M reductase system.

Appearance of uridine 5'-diphospho-N-acetylglucosamine-4-epimerase during sporulation of Bacillus megaterium.

In a biosynthetic study of the spore coat of Bacillus megaterium ATCC 12872 spore with galactosamine phosphate as a major component of the outer coat, high-performance liquid chromatography (HPLC) and enzyme immunoassay were applied for the measurement of UDP-N-acetylglucosamine-4-epimerase [EC 5.1.3.7] activity and the enzyme protein concentration, respectively. The new HPLC system using an ion-pair (or anion-exchange) column allowed us to determine successfully the enzyme activity and its application, proving that the specific activity of the enzyme in the cells increased at the later stage of sporulation. This increase in activity was parallel to the induction of enzyme protein synthesis, which was detected by sandwich enzyme immunoassay using antiserum to the purified enzyme. These results suggested that the regulation of this enzyme is at the genetic level and it plays an important role in the outer coat synthesis in the later sporulation stage of B. megaterium.

A simplified procedure for synthesizing large quantities of highly purified uridine [beta-32P]diphospho-N-acetylglucosamine.

A simplified procedure for the synthesis of [beta-32P]UDP-N-acetylglucosamine is described. This novel method utilizes commercially available enzymes, chemical acetylation, and preparative thin-layer chromatography and can be used to synthesize millicurie quantities of the sugar nucleotide at an extremely high specific activity and purity.

Pool levels of UDP N-acetylglucosamine and UDP N-acetylglucosamine-enolpyruvate in Escherichia coli and correlation with peptidoglycan synthesis.

A high-pressure liquid chromatography procedure was developed for the isolation and quantitation of UDP-N-acetylglucosamine, UDP-N-acetylglucosamine-enolpyruvate, and UDP-N-acetylmuramic acid, which are the early cytoplasmic precursors of bacterial peptidoglycan. In exponential-phase cells of Escherichia coli K-12, the intracellular concentration of UDP-N-acetylglucosamine was about 100 microM, whereas that of UDP-N-acetylglucosamine-enolpyruvate was only 2 microM. The phosphoenolpyruvate: UDP-N-acetylglucosamine transferase and UDP-N-acetylglucosamine-enolpyruvate reductase activities were investigated in extracts from E. coli. These activities appeared to be present in amounts sufficient for the ongoing rate of peptidoglycan synthesis. Certain uridine nucleotide peptidoglycan precursors were found to inhibit phosphoenolpyruvate: UDP-N-acetylglucosamine transferase activity.

The enzymic synthesis of beta-[32P]UDP-N-acetylglucosamine.

Cells of Micrococcus sp. 2102 incorporate inorganic [32P]phosphate from the medium into the sugar-phosphate polymer of the wall. Controlled acid hydrolysis of sodium dodecyl sulphate-extracted cells gives N-acetylglucosamine 6-[32P]-phosphate which can be purified by ion-exchange chromatography and incubated with UTP in the presence of crude preparations of phosphoacetylglucosamine mutase from Neurospora crassa and UTP:N-acetylglucosamine 1-phosphate phosphotransferase from Bacillus licheniformis which act in concert to synthesise beta-[32P]UDP-N-acetylglucosamine.