Biological synthesis of quercetin 3-<Emphasis Type="Italic">O</Emphasis>-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine conjugate using engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing <Emphasis Type="Italic">UGT78D2</Emphasis>

Biotransformation of flavonoids using Escherichia coli harboring nucleotide sugar-dependent uridine diphosphate-dependent glycosyltransferases (UGTs) commonly results in the production of a glucose conjugate because most UGTs are specific for UDP-glucose. The Arabidopsis enzyme AtUGT78D2 prefers UDP-glucose as a sugar donor and quercetin as a sugar acceptor. However, in vitro, AtUGT78D2 could use UDP-N-acetylglucosamine as a sugar donor, and whole cell biotransformation of quercetin using E. coli harboring AtUGT78D2 produced quercetin 3-O-N-acetylglucosamine. In order to increase the production of quercetin 3-O-N-acetylglucosamine via biotransformation, two E. coli mutant strains deleted in phosphoglucomutase (pgm) or glucose-1-phosphate uridylyltransferase (galU) were created. The galU mutant produced up to threefold more quercetin 3-O-N-acetylglucosamine than wild type, resulting in the production of 380-mg/l quercetin 3-O-N-acetylglucosamine and a negligible amount of quercetin 3-O-glucoside. These results show that construction of bacterial strains for the synthesis of unnatural flavonoid glycosides is possible through rational selection of the nucleotide sugar-dependent glycosyltransferase and engineering of the nucleotide sugar metabolic pathway in the host strain.     

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine metabolism in <Emphasis Type="Italic">Bifidobacterium</Emphasis>

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism. S. Foley and E. Stolarczyk contributed equally to this work     

Synthesis of five nona-β-(1→6)-d-glucosamines with various patterns of N-acetylation corresponding to the fragments of exopolysaccharide of Staphylococcus aureus

A series of five 3-acetamidopropyl β-glycosides of nona-β-(1→6)-glucosamines containing two N-acetylglucosamine residues separated by a different number of glucosamine units with free amino groups have been synthesized using a convergent blockwise approach. Oxazoline glycosylation was used to introduce N-acetylglucosamine residues. These nonasaccharides are structurally related to the poly-N-acetylglucosamine (PNAG) extracellular polysaccharide of Staphylococcus aureus and can be used as models for biochemical and immunological studies.     

PURIFICATION AND PROPERTIES OF BRAIN N-ACETYL-β-HEXOSAMINIDASE A

—N-Acetyl-β-hexosaminidase A was purified to homogeneity from human and monkey brains by the conventional procedures followed by concanavalin A–Sepharose affinity chromatography. The optimal activity was observed at pH 4·5 for both enzyme preparations with both the aglycones N-acetylglucosamine and N-acetylgalactosamine derivatives. The K_m values for hexosaminidase A from monkey brain were 0·26 mm and 0·04 mm respectively for N-acetylglucosamine and N-acetylgalactosamine. K_m values obtained for glucosamine and galactosamine derivatives for the human brain hexosaminidase A were of the same order. The glycoprotein nature of the enzymes was established by the affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the enzyme molecule from monkey brain.     

EFFICIENT PRODUCTION OF URIDINE 5′-DIPHOSPHO-N-ACETYLGLUCOSAMINE BY THE COMBINATION OF THREE RECOMBINANT ENZYMES AND YEAST CELLS

Uridine 5′-diphospho N-acetylglucosamine (UDP-GlcNAc) is an important nucleotide sugar in the biochemistry of all living organisms, and it is an important substrate in the synthesis of oligosaccharides. In the present work, three bioactive enzymes, namely, glucokinase (YqgR), GlcNAc-phosphate mutase (Agm1), and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), were produced effectively as soluble form in recombinant Escherichia coli. These three enzymes and dried yeast together were used to construct a multistep enzymatic system, which could produce UDP-GlcNAc efficiently with N-acetylglucosamine (GlcNAc) as the substrate. After the optimization of various reaction conditions, 31.5 mMUDP-GlcNAc was produced from 50 mMGlcNAc and 50 mMUMP.     

Fertilization studies in the hamster : The role of cell-surface carbohydrates

The nature of complementary binding sites on the surfaces of hamster gametes has been analysed using mono- and oligosaccharides, glycoproteins and glycosidases in an in vitro system. The binding of capacitated spermatozoa to the zona pellucida was inhibited by several mono- and oligosaccharides related to fucose, galactose, and acetylated amino sugars, but not by unrelated sugars. Several glycoproteins with prosthetic carbohydrate groups rich in or terminated by galactose or N-acetylglucosamine residues were also potent inhibitors of fertilization. Of all the glucoproteins tested, two plasma glycoproteins, α₁-acid glycoproteins (orosomucoid) and fetuin were most effective. In their native form they were non-inhibitory but their desialylated (galactoseterminated) forms completely prevented the sperm-zona binding. Agalacto-orosomucoid with N-acetylglucosamine terminals also inhibited fertilization. The treatment of capacitated spermatozoa with α- -fucosidase, α- -galactosidase and β-N-acetylhexosaminidase, but not with other glycosidases, trypsin and arylsulphatase, resulted in the complete inhibition of fertilization. Inhibitory saccharides and glycosidases did not interfere with sperm motility and had no effect on sperm-oolemma fusion. The pretreatment of cumulus-free oocytes with these agents did not inhibit sperm zona pellucida binding either. These results provide evidence that sperm-zona pellucida binding is mediated by ligands on the sperm surface containing fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine residues.     

Separation of dolichylpyrophosphoryloligosaccharides by liquid chromatography

Fourteen dolichylpyrophosphoryloligosaccharides, precursors of the asparagine-linked oligosaccharides of glycoproteins, have been separated by liquid chromatography on silica gel. The dolichylpyrophosphoryl-N-acetylglucosamine and the dolichylpyrophosphoryl-(N-acetylglucosamine)₂-(mannose)₉(glucose)_2,3 thus resolved were shown to retain their activity as substrates in enzyme catalyzed reactions. The chromatography procedure for the first time makes available many of these single intermediates for further study.     

A Fluorescence-Based Homogeneous Assay for Measuring Activity of UDP–3-O-(R-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase

UDP–3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is one of the key enzymes of bacterial lipid A biosynthesis, catalyzing the removal of the N-acetyl group of UDP–3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. The lpxC gene is essential in Gram-negative bacteria but absent from mammalian genomes, making it an attractive target for antibacterial drug discovery. Current assay methods for LpxC are not suitable for high throughput screening, since they require multiple product separation steps and the use of radioactively labeled material that is difficult to prepare. A homogenous fluorescence-based assay was developed that uses UDP–3-O-(N-hexyl-propionamide)-N-acetylglucosamine as a surrogate substrate. This surrogate can be prepared from commercially available UDP–GlcNAc by enzymatic conversion to UDP–MurNAc, which is then chemically coupled to n-hexylamine. Following the LpxC reaction, the free amine of the deacetylation product can be derivatized by fluorescamine, thus generating a fluorescent signal. This surrogate substrate has a K_m of 367 μM and k_cat of 0.36 s⁻¹, compared to 2 μM and 1.5 s⁻¹ for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC₅₀s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Systematic synthesis and inhibitory activity of haloacetamidyl oligosaccharide derivatives toward cytoplasmic peptide:<Emphasis Type="Italic">N</Emphasis>-glycanase

A series of glycosyl haloacetamides were synthesized as potential inhibitors of cytoplasmic peptide:N-glycanase (PNGase), an enzyme that removes N-glycans from misfolded glycoproteins. Chloro-, bromo-, and iodoacetamidyl chitobiose and chitotetraose derivatives exhibited a significant inhibitory activity. No inhibitory activity was observed with of fluoroacetamididyl derivatives. Moreover, N-acetylglucosamine derivatives, β-chloropropionamidyl chitobiose, and chloroacetamidyl cellooligosaccharide derivatives did not show any activity. These results underscore the importance of the N-acetyl groups of chitobiose for PNGase recognition. In addition, reactivity and position of the leaving group at the reducing end are also important factors.     

UDP-Gal: <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine β 1–4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.     

Extracellular,low-affinity β-N-acetylglucosaminidases linked to the dynamics of diatoms and crustaceans in freshwater systems of different trophic degree

Extracellular hydrolysis of 4-methylumbelliferyl β-N-acetylglucosaminide was measured in the oligomesotrophic Piburger See and the eutrophic Římov reservoir during spring and summer phytoplankton blooms, respectively. Total enzymatic activity (TEA) ranged between 0.2 and 19.1 nmol 1⁻¹ h⁻¹ in the reservoir and between 0.8 and 12.4 nmol 1⁻¹ h⁻¹ in the lake. High-affinity (K_m < 1 μmol 1⁻¹) and lowaffinity (K_m > 100 μmol 1⁻¹) enzymes were kinetically identifiable in most samples from both localities. The low-affinity enzyme activity (LEA) usually accounted for >60% (mean: 80%) of TEA. LEA and diatom biomass significantly correlated over time in the reservoir epilimnion (r_s = 0.578) and in the lake metalimnion (r_s = 0.862). As diatoms possess chitin and take up its monomer, N-acetylglucosamine, two explanations of the observed relationships are suggested: extracellular β-N-acetylglucosaminidase activity partly originates either from ectoenzymes of chitinolytic bacteria attached to diatom cells or from ectoenzymes of diatoms, enabling them to take up N-acetylglucosamine from ambient amino sugars instead of synthesizing it de novo. A significant positive correlation of LEA with crustacean abundance was found in the lake epilimnion (r_s = 0.850), apparently reflecting the growing spring populations of frequently moulting juvenile crustaceans. A possible contribution of chitinolytic bacteria, accompanying the crustacean populations, to LEA is discussed.     

Increased bisecting and core-fucosylated <Emphasis Type="Italic">N</Emphasis>-glycans on mutant human amyloid precursor proteins

Alteration of glycoprotein glycans often changes various properties of the glycoprotein. To understand the significance of N-glycosylation in the pathogenesis of early-onset familial Alzheimer’s disease (AD) and in β-amyloid (Aβ) production, we examined whether the mutations in the amyloid precursor protein (APP) gene found in familial AD affect the N-glycans on APP. We purified the secreted forms of wild-type and mutant human APPs (both the Swedish type and the London type) produced by transfected C17 cells and determined the N-glycan structures of these three recombinant APPs. Although the major N-glycan species of the three APPs were similar, both mutant APPs contained higher contents of bisecting N-acetylglucosamine and core-fucose residues as compared to wild-type APP. These results demonstrate that familial AD mutations in the polypeptide backbone of APP can affect processing of the attached N-glycans; however, whether these changes in N-glycosylation affect Aβ production remains to be established. Keiko Akasaka-Manya and Hiroshi Manya contributed equally to this work.     

Novel products in hyaluronan digested by bovine testicular hyaluronidase

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.     

Subcellular characterization of glycoproteins in the principal cells of human gallbladder

Gallbladder mucus is mainly composed of glycoproteins, which seem to play a critical role in cholesterol nucleation during gallstone formation. The biosynthetic pathway and sequential processing as well as the characterization of the oligosaccharide sidechains of human gallbladder secretory glycoproteins have not been completely defined. The aim of the present study is the subcellular characterization of the glycoproteins in the principal cells of human gallbladder. Principal cells of normal human gallbladder were studied by means of a variety of cytochemical techniques, including lectin histochemistry, enzyme and chemical treatments, immunocytochemistry and lectin-gold technology. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid residues were detected in mucous granules, Golgi apparatus and apical membrane of principal cells. Mannose residues were only observed in dense bodies. Oligosaccharide side-chains of the glycoproteins contained in the biliary mucus are synthesized in the Golgi apparatus of the principal cells of the gallbladder epithelium and are also contained in the mucous granules of these cells. Terminal N-acetylneuraminic acid(2-3)galactose(1-3)N-acetylgalactosamine, N-acetylneuraminic acid(2-3)galactose(1-4)N-acetylglucosamine and galactose(1-4)N-acetylglucosamine sequences are contained in the oligosaccharide chains of gallbladder mucus glycoproteins. The dense bodies detected in the cytoplasm of the principal cells contained N-linked glycoproteins. Mucin-type O-linked glycoproteins were the main components of the mucous granules although some N-linked chains were also detected.     

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I gene

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.     

Alteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction

Expanding the scope of stereoselectivity is of current interest in enzyme catalysis. In this study, using error-prone polymerase chain reaction (PCR), a thermostable adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) from Thermus caldophilus GK-24 has been altered to improve its catalytic activity toward enatiomeric substrates including [glucose-1-phosphate (G-1-P) + uridine triphosphate (UTP)] and [N-acetylglucosamine-1-phosphate (GlcNAc) + UTP] to produce uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine, respectively. To elucidate the amino acids responsible for catalytic activity, screening for UDP-glucose pyrophosphorylase (UGPase) and UDP-N-acetylglucosamine pyrophosphorylase (UNGPase) activities was carried out. Among 656 colonies, two colonies showed UGPase activities and three colonies for UNGPase activities. DNA sequence analyses and enzyme assays showed that two mutant clones (H145G) specifically have an UGPase activity, indicating that the changed glycine residue from histidine has the base specificity for UTP. Also, three double mutants (H145G/A325V) showed a UNGPase, and A325 was associated with sugar binding, conferring the specificity for the sugar substrates and V325 of the mutant appears to be indirectly involved in the binding of the N-acetylamine group of N-acetylglucosmine-1-phosphate. The authors Hosung Sohn and Yong-Sam Kim equally contributed to the study.     

Resistance to nodulation of cv. Afghanistan peas is overcome by nodX, which mediates an O-acetylation of the Rhizobium leguminosarum lipo-oligosaccharide nodulation factor

Only some strains of Rhizobium leguminosarum biovar viciae can efficiently nodulate varieties of peas such as cv. Afghanistan, which carry a recessive allele that blocks efficient nodulation by most western isolates of R. I. viciae. One strain (TOM) which can nodulate cv. Afghanistan peas has a gene (nodX) that is required to overcome the nodulation resistance. Strain TOM makes significantly lower amounts of lipo-oligosaccharide nodulation factors than other strains of R. I. viciae. and this effect appears to be due to lower levels of nod gene induction. These nodulation factors are similar to those from other R. I. viciae. strains in that they consist of an oligomer of four or five β1-4-linked N-acetylglucosamine residues in which the terminal non-reducing glucosamine carries an O-acetyl group and a C_18:4 or C_18:1N-acyl group. However, one of the nodulation factors made by strain TOM differs from the factors made by other strains of R. I. viciae. in that it carries an O-acetyl group on the C-6 of the reducing N-acetylglucosamine residue. This acetylation is NodX-dependent and the pentameric nodulation factor is acetylated on the reducing N-acetylglucosamine residue whereas the tetrameric nodulation factor is not. Although the nodL gene product is also an O-acetyl transferase (it O-acetylates the C-6 of the terminal non-reducing glucosamine), there is very little similarity between the amino acid sequences of these two acetyl transferases.     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Transacetylation of carbohydrates in organic solvent catalysed by O-acetylpeptidoglycan esterase from Neisseria gonorrhoeae

The potential of the Neisseria gonorrhoeae O-acetylpeptidoglycan esterase (Ape1a) for catalysing transacetylations in organic solvents with a number of carbohydrate acceptors was investigated. The performance of the enzyme was observed to improve as the polarity index of the solvent increased. The best transacetylation conditions were determined to be a 1:6 phosphate buffer/ethyl acetate system, where Ape1a catalysed approximately 28% acetylation of 4-methylumbelliferyl-N-acetylglucosamine using p-nitrophenyl acetate as donor. Further analysis of the acetylated products by reverse phase HPLC and ESI-mass spectrometry confirmed the presence of monoacetylated 4-methylumbelliferyl-N-acetylglucosamine. Under identical reaction conditions, the enzyme also performed transacetylations using ethyl acetate or vinyl acetate as donor. These results demonstrated the feasibility of using the bacterial cell wall enzyme Ape1a to generate hitherto unattainable compounds which may be used as antagonists of peptidoglycan-metabolizing enzymes.