The mechanism of biosynthesis and direction of chain extension of a polyl-(N-acetylglucosamine 1-phosphate) from the walls of Staphylococcus lactis N.C.T.C. 2102

1. The synthesis of a polymer of N-acetylglucosamine 1-phosphate, occurring in the walls of Staphylococcus lactis N.C.T.C. 2102, was examined by using cell-free enzyme preparations. The enzyme system was particulate, and probably represents fragmented cytoplasmic membrane. 2. Uridine diphosphate N-acetylglucosamine was the only substrate required for polymer synthesis and labelled substrate was used to show that N-acetylglucosamine 1-phosphate is transferred as an intact unit from substrate to polymer. 3. The properties of the enzyme system were studied. A high concentration of Mg(2+) or Mn(2+) was required for optimum activity, and the pH optimum was about 8.5. 4. End-group analysis during synthesis in vitro showed that newly formed chains contain up to about 15 repeating units. Pulse-labelling indicated that chain extension occurs by transfer from the nucleotide to the ;sugar-end' of the chain, i.e. to the end that is not attached to peptidoglycan in the wall.     

Induction of N-acetylmannosamine catabolic pathway in yeast.

N-Acetylmannosamine kinase activity is absent from yeast cells grown on N-acetylmannosamine. However, other enzymes of the catabolic pathway, namely, N-acetylmannosamine-2-epimerase, N-acetylglucosamine kinase and glucosamine-6-phosphate deaminase are induced. In addition, a high affinity uptake system (permease) for the uptake of N-acetylglucosamine is synthesized under these conditions. The presence of either N-acetylmannosamine or N-acetylglucosamine as inducer is essential for the induced synthesis of these enzymes. The enzyme synthesis stops and their concentration in the cells declines rapidly as soon as inducer is removed from the medium. N-Acetyl-D-galactosamine can also induce all these enzymes except for N-acetylmannosamine-2-epimerase, suggesting the convergence of catabolic pathways for both the aminosugars at N-acetyl-D-glycosamine. Experiments with inhibitors of macromolecule synthesis suggest that the snythesis of RNA and protein is necessary for the induction of these cyzymes whereas the synthesis of DNA is not.     

A lipid intermediate in the synthesis of a poly-(N-acetylglucosamine 1-phosphate) from the wall of Staphylococcus lactis N.C.T.C. 2102

1. The enzymic synthesis of the wall polymer poly-(N-acetylglucosamine 1-phosphate) in Staphylococcus lactis N.C.T.C. 2102 was studied by using UDP-[acetyl-(14)C]N-acetylglucosamine and the corresponding nucleotide containing (32)P. 2. Labelled material was extracted from the particulate enzyme preparation with butan-1-ol. Pulse-labelling experiments indicated that this material contained an intermediate in the biosynthesis. 3. The lipid intermediate was partially purified, and chemical and enzymic degradation showed that it was composed of N-acetylglucosamine 1-pyrophosphate in labile ester linkage to an organic-soluble alcohol, possibly a polyisoprenoid alcohol. The methanolysis of sugar 1-pyrophosphate derivatives, including nucleoside diphosphate sugars, is discussed in relation to degradation products obtained from the lipid. 4. The lipids from the particulate enzyme preparation probably contained another compound in which N-acetylglucosamine 1-phosphate is attached to an organic-soluble alcohol; this may participate in the biosynthesis of another polysaccharide. 5. The function of the lipid intermediate in polymer biosynthesis is discussed.     

Two distinct UDP-galactose: 2-acetamido-2-deoxy-D-glucose 4 beta-galactosyltransferases in porcine trachea

In previous studies on glycosyltransferase activities in porcine trachea, we demonstrated the presence of two galactosyltransferases which transfer galactose from UDP-galactose to N-acetylglucosamine (Sheares, B.T. and Carlson, D.M. (1983) J. Biol. Chem. 258, 9893-9898). One enzyme, UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase, synthesized galactosyl-beta 1,3-N-acetylglucosamine while the other, UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase, synthesized galactosyl-beta 1,4-N-acetylglucosamine. A third galactosyltransferase has now been demonstrated utilizing a solubilized membrane preparation from pig trachea, which also synthesizes galactosyl-beta 1,4-N-acetylglucosamine as determined by gas-liquid chromatography and Diplococcus pneumoniae beta-galactosidase treatment. This new UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase is distinct from the lactose synthetase A protein in that it does not bind to alpha-lactalbumin-agarose or to N-acetylglucosamine-agarose. The enzyme is separable from the UDP-galactose:N-acetylgalactosaminyl-mucin 3 beta-galactosyltransferase by affinity chromatography on asialo ovine submaxillary mucin adsorbed to DEAE-Sephacel. This newly discovered 4 beta-galactosyltransferase binds to UDP-hexanolamine-Sepharose and is partially separated from UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase by Sephacryl S-200 gel filtration chromatography. Neither high concentrations of N-acetylglucosamine (200 mM) nor alpha-lactalbumin inhibits the incorporation of galactose into galactosyl-beta 1,4-N-acetylglucosamine by this enzyme.     

Lipid intermediates in the biosynthesis of the wall teichoic acid in Staphylococcus lactis I3

1. Particulate enzyme systems have been prepared from Staphylococcus lactis I3 which effect the synthesis of wall teichoic acid (a polymer containing a repeating unit in which d-glycerol 1-phosphate is attached to the 4-position on N-acetylglucosamine 1-phosphate) from the nucleotide precursors CDP-glycerol and UDP-N-acetylglucosamine. By using nucleotides labelled with (32)P and (14)C it has been shown that the synthesis proceeds via lipid intermediates. 2. Two intermediates have been found. In one of these N-acetylglucosamine 1-phosphate is present, whereas in the other the repeating unit of the teichoic acid occurs. 3. The simultaneous formation of the teichoic acid, a poly-(N-acetylglucosamine 1-phosphate) and an unidentified lipid, together with the poor ability of most particulate systems to synthesize polymer and the instability of the lipid intermediates themselves, have interfered with pulse-labelling experiments. Nevertheless, the biosynthetic sequence has been elucidated. It is concluded that the intermediates are derivatives of undecaprenol phosphate.     

Biosynthesis of galactosyl-beta 1,3-N-acetylglucosamine

The biosynthesis of galactosyl-beta 1,3-N-acetylglucosamine has been demonstrated using membrane preparations from pig trachea. Unlike the UDP-galactose:2-acetamido-2-deoxy-D-glucose 4 beta-galactosyltransferase, which is inhibited by high levels of N-acetylglucosamine, the UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase shows no inhibition at 200 mM N-acetylglucosamine. About 80% of the total disaccharide synthesized at 200 mM N-acetylglucosamine was base-labile suggesting the 1,3-linkage, alpha-Lactalbumin inhibits galactose incorporation into galactosyl-beta 1,4-N-acetylglucosamine but has little or no effect on the activity of the 1,3-galactosyltransferase. Escherichia coli beta-galactosidase readily hydrolyzed the base-stable product, but not the base-labile component. The apparent 1,3-linked disaccharide was reduced with NaBH4 and was isolated by Bio-Gel P-2 column chromatography. Methylation analysis by gas chromatography/mass spectrometry showed tetramethyl galactose and a 3-substituted N-acetylglucosaminitol. Neither the beta 1,4 nor the beta 1,3 disaccharide was hydrolyzed by green coffee bean alpha-galactosidase. Both disaccharides were readily hydrolyzed by bovine testes beta-galactosidase. This is the first report on the galactosyltransferase which catalyzes the synthesis of the galactosyl-beta 1,3-N-acetylglucosamine linkage such as found in the Type I chain of human blood group substances. A tissue survey in rats showed only rat intestine to have readily detectable UDP-galactose: N-acetylglucosamine 3 beta-galactosyltransferase activity. The intestinal membrane fraction like the tracheal enzyme catalyzes the synthesis of two disaccharides as judged by base treatment, and these appear to be the beta 1,3 and beta 1,4 isomers of galactosyl-N-acetylglucosamine.     

Regulation of chitinase synthesis in Trichoderma harzianum.

The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0.5%, at 28 degrees C, pH 6.0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.     

Synthesis and proteolytic activation of chitin synthetase in Phycomyces blakesleeanus Burgeff

The chitin synthetase of Phycomyces blakesleeanus mycelium is a particulate enzyme sedimenting mostly at 1000xg. The activity in crude extracts or cellular fractions can be increased more than tenfold by mild trypsin treatment. Plotting the reaction velocity versus UDP-N-acetylglucosamine concentration yields a sigmoidal curve. N-acetylglucosamine, which greatly stimulates the enzyme, changes the kinetics to an almost normal hyperbolic relationship.The enzyme is nearly absent in dormant spores and is synthesized de novo in germinating spores (from 4 h germination on). Trypsin treatment of extracts from germinating spores to assay the synthesis of the proenzyme did not reveal an earlier synthesis of the zymogen, which therefore might have some activity of its own.Abbreviations Used UDP-GlcNAc Uridinediphosphate-N-acetylglucosamine - GlcNAc N-acetylglucosamine - Chitin synthetase UDP-2-acetylamino-deoxyglucosyltransferase (EC 2.4.1.16)     

Molecular cloning and characterization of murine and human N-acetylglucosamine kinase.

N-Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino-acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N-acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N-acetylglucosamine kinase activity as well as N-acetylmannosamine kinase activity. The complete cDNA sequence of human N-acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N-acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino-acid level. Amino-acid sequences of murine and human N-acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present. Tissue distribution of murine N-acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.     

Regulation of N-acetylglucosamine uptake in yeast.

Various yeasts have been investigated for their ability to grow on N-acetylglucosamine as the sole carbon source and only those which are associated with the disease, candidiasis, gave positive results. The yeasts unable to grow on N-acetylglucosamine lacked the capacity to transport the aminosugar across the cell membrane. In pathogenic yeasts, two systems of different affinity for substrate were found to operate in the uptake of N-acetylglucosamine. In glucose-grown cells a constitutive, low affinity uptake system was present, but upon addition of inducer, a specific high affinity uptake system was synthesized. Experiments with the inhibitors of macromolecule synthesis suggested that the synthesis of RNA and protein is necessary for induction whereas the synthesis of DNA is not. In glucose-grown Candida albicans cells which are devoid of N-acetylglucosamine enters into the cells as phosphorylated form using a constitutive uptake system. Uranyl acetate (0.01 mM) which binds to cell membrane-associated polyphosphates, inhibited completely the inducible uptake of N-acetylglucosamine. Labelling experiments, designed to determine the temporal sequence of appearance of N-acetylglucosamine in intracellular free sugar and sugar-phosphate pools, indicated that N-acetylglucosamine first appeared in the cells as pohosphorylated form. Similar results were obtained with Saccharomyces phosphorylated form. Similar results were obtained with Saccharomyces cerevisiae 3059 and some other yeasts which are devoid of N-acetylglucosamine kinase in both uninduced and induced conditions. These results are consistent with the model of van Steveninck that involves phosphorylation during transpost. Furthermore, inhibitors of energy metabolism (arsenate, azide and cyanide), proton conductor (m-chlorocarbonylcyanide phenylhydrazine) and dibenzyl diammonium ion (membrane permeable cation) inhibited the inducible N-acetylglucosamine uptake in C. albicans.     

An enzyme catalyzing the liberation of N-acetylglucosamine from N-acetylglucosaminyl pyrophosphorylpolyprenol in Bacillus polymyxa membranes

A novel enzyme which specifically hydrolyzes N-acetylglucosaminyl pyrophosphorylpolyprenol to liberate N-acetylglucosamine was found in membranes of Bacillus polymyxa AHU 1385. The enzyme seems to be inactive toward alpha-N-acetylglucosaminyl phosphorylundecaprenol, beta-N-acetylglucosaminyl phosphorylundecaprenol, N-acetylglucosamine 1-phosphate, N-acetylglucosamine 1-pyrophosphate, or UDP-N-acetylglucosamine. Much lower activities of the same enzyme were also found in membranes of several other strains of Bacilli.     

Catabolism of glycoprotein glycans. Characterization of a lysosomal endo-N-acetyl-beta-D-glucosaminidase specific for glycans with a terminal chitobiose residue

An endo-N-acetyl-beta-D-glucosaminidase active towards oligosaccharides with a reducing terminal [bis(N-acetylglucosamine)]residue has been characterized in rat liver. The primary structure of its reaction products was determined using high-resolution 1H-NMR spectroscopy. The enzyme is predominantly located in the lysosomal fraction, presents a maximum of activity at pH 3.5 and is completely inactive towards conjugated glycans, i.e. glycoproteins and glycopeptides as well as on glycoasparagines. These results support the existence of a new pathway for the degradation of glycoprotein glycans inside the lysosome. In particular, this enzymic activity may be the origin of oligosaccharides bearing a single terminal reducing N-acetylglucosamine residue which are excreted in the urine of patients with various exoglycosidase deficiencies.     

The cellular basis of chitin synthesis in fungi and insects: common principles and differences

Chitin is a polymer of N-acetylglucosamine, which assembles into microfibrils of about 20 sugar chains. These microfibrils serve as a structural component of natural biocomposites found in cell walls and specialized extracellular matrices such as cuticles and peritrophic membranes. Chitin synthesis is performed by a wide range of organisms including fungi and insects. The underlying biosynthetic machinery is highly conserved and involves several enzymes, of which the chitin synthase is the key enzyme. This membrane integral glycosyltransferase catalyzes the polymerization reaction. Most of what we know about chitin synthesis derives from studies of fungal and insect systems. In this review, common principles and differences will be worked out at the levels of gene organization, enzymatic properties, cellular localization and regulation.     

Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors

We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (beta 4-glucuronic acid-beta 3-N-acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (approximately 10-20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (approximately 1-100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicant-tagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.     

A pathway of chitosan formation in Mucor rouxii: enzymatic deacetylation of chitin

An enzyme which hydrolyzes the acetamido groups of N-acetylglucosamine residues in chitin was partially purified from $\text{Mucor rouxii}$. The enzyme deacetylates also N-acetylchitooligoses, whereas it is inactive toward bacterial cell wall peptidoglycan, N-acetylated heparin, a polymer of N-acetylgalactosamine, di-N-acetylchitobiose, or N-acetylglucosamine. The enzyme shows a pH optimum of 5.5 and is markedly inhibited by acetate. The occurrence of this enzyme accounts for the formation of chitosan in fungi.     

Structure and function of N-acetylglucosamine kinase. Identification of two active site cysteines.

N-Acetylglucosamine is a major component of complex carbohydrates. The mammalian salvage pathway of N-acetylglucosamine recruitment from glycoconjugate degradation or nutritional sources starts with phosphorylation by N-acetylglucosamine kinase. In this study we describe the identification of two active site cysteines of the sugar kinase by site-directed mutagenesis and computer-based structure prediction. Murine N-acetylglucosamine kinase contains six cysteine residues, all of which were mutated to serine residues. The strongest reduction of enzyme activity was found for the mutant C131S, followed by C143S. Determination of the kinetic properties of the cysteine mutants showed that the decreased enzyme activities were due to a strongly decreased affinity to either N-acetylglucosamine for C131S, or ATP for C143S. A secondary structure prediction of N-acetylglucosamine kinase showed a high homology to glucokinase. A model of the three-dimensional structure of N-acetylglucosamine kinase based on the known structure of glucokinase was therefore generated. This model confirmed that both cysteines are located in the active site of N-acetylglucosamine kinase with a potential role in the binding of the transferred gamma-phosphate group of ATP within the catalytic mechanism.     

尿苷二磷酸糖固定化酶合成法研究

利用生物酶进行体外催化反应合成不同种类的尿苷二磷酸糖（uridine diphosphate sugar,UDP-糖）,生物酶的重复利用率较低。为提高尿苷二磷酸糖的合成效率及增加产物种类,以镍螯合聚丙烯酸酯树脂为载体,对带有HIS标签的N-乙酰己糖胺激酶（N-acetylhexosamine kinase,NahK）和尿苷转移酶（uridine transferase,GlmU）进行固定化。以固定化NahK和固定化GlmU为催化酶,不同单糖作为底物,研究尿苷二磷酸糖的一锅法合成情况。利用Q柱对产物进行纯化,通过高效液相色谱法、质谱法、核磁共振氢谱法对反应产物进行检测。确定了镍螯合聚丙烯酸酯树脂对游离NahK和GlmU的实际载量分别为10和20 mg·g^-1。固定化酶量的最优配比为5.5 g固定化NahK和2.5 g固定化GlmU。固定化酶的最适pH和温度分别为8.0和35℃,且能在重复反应中稳定反应5个批次。葡萄糖、N-乙酰氨基葡萄糖和甘露糖可以参与一锅法反应,生成UDP-糖的相对分子质量分别为566、607、566,而葡萄糖醛酸、半乳糖和果糖在该体系下不能合成相应的UDP-糖。基于固定化酶技术,一锅法可合成UDP-葡萄糖、UDP-N-乙酰氨基葡萄糖、UDP-甘露糖。     

Evidence that the lipid carrier for N-acetylglucosamine is different from that for mannose in mung beans and cotton fibers

Cell-free enzyme particles from mung beans (Phaseolus aureus) or cotton (Gossypium hirsutum L.) fibers catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and N-acetylglucosamine from UDP-[³H]-N-acetylglucosamine into polyprenyl-type lipids. These lipids have been synthesized and purified and the lipid moieties compared to each other as well as to dolichyl phosphate and to lipids isolated from similar mannoseand N-acetylglucosamine-containing lipids from liver and aorta.

The following lines of evidence indicate that in plants, the lipid carrier for N-acetylglucosamine is different from the lipid carrier for mannose: [List: see text]

We propose that the apparent difference in the lipid carrier for these two sugars may be a point of control of glycoprotein synthesis.