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PubMed1998

Cloning and sequence analysis of Histoplasma capsulatum glucosamine-6-phosphate synthase gene fragment.

The 3' part of the glucosamine-6-phosphate synthase gene from Histoplasma capsulatum was PCR amplified using degenerate primers designed from the known glucosamine-6-phosphate synthase gene sequences, cloned and sequenced. The computer analysis of the 676 bp sequence revealed the presence of two introns. The identities of the deduced amino acid sequence to the corresponding Saccharomyces cerevisiae and Candida albicans fragment are 65 and 63.8%, respectively.

Amino Acid Sequence↗
PubMed2006

Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme.

The glmS ribozyme resides in the 5' untranslated region of glmS mRNA and functions as a catalytic riboswitch that regulates amino sugar metabolism in certain Gram-positive bacteria. The ribozyme catalyzes self-cleavage of the mRNA and ultimately inhibits gene expression in response to binding of glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein. We have used nucleotide analog interference mapping (NAIM) and suppression (NAIS) to investigate backbone and nucleobase functional groups essential for ligand-dependent ribozyme function. NAIM using GlcN6P as ligand identified requisite structural features and potential sites of ligand and/or metal ion interaction, whereas NAIS using glucosamine as ligand analog revealed those sites that orchestrate recognition of ligand phosphate. These studies demonstrate that the ligand-binding site lies in close proximity to the cleavage site in an emerging model of ribozyme structure that supports a role for ligand within the catalytic core.

Bacterial Proteins↗
PubMed1992

Human glucosamine-6-sulphatase deficiency. Diagnostic enzymology towards heparin-derived trisaccharide substrates.

Glucosamine-6-sulphatase (6S) activity towards a series of radiolabelled heparin-derived trisaccharide substrates was determined in cultured human skin fibroblast and leucocyte homogenates, and in urine supernatants of normal individuals and patients affected with 6S deficiency [Sanfilippo D syndrome; mucopolysaccharidosis (MPS) type IIID]. The N-sulphated and N-acetylated derivatives of the trisaccharide substrate O-(alpha-glucosamine 6-sulphate)-(1----4)-L-O-(alpha-iduronic acid 2-sulphate)-(1----4)-D-O-2,5-anhydro[1-3H]mannitol 6-sulphate (GlcNH6S-IdoA2S-anM6S) were prepared by enzymic digestion of a pentasulphated tetrasaccharide isolated following the HNO2 deamination of heparin. Purified lysosomal enzymes and MPS-patient skin fibroblasts were used along with chemical degradation to confirm the structure of each of the substrates that were utilized to study the interaction of the enzyme activities required to degrade the highly sulphated regions of heparan sulphate. Human liver, skin fibroblast and urine 6S activities were separated by chromatofocusing into at least four and possibly up to six individual activities. 6S activities present in each of the tissues generally had similar catalytic properties, including Km values, pH optima and inhibition with NaCl, Na2SO4 and NaH2PO4. Leucocyte and skin fibroblast 6S activities towards GlcNAc6S-IdoA2S-anM6S were maximal at pH 4.1 and 3.9 respectively, with Km values of 2.8 microM and 0.9-1.7 microM respectively. Urine 6S activity towards GlcNAc6S-IdoA2S-anM6S was stimulated 30-fold by BSA at pH 3.9, which shifted the pH optimum from 5.1 to 4.2 and decreased the Km value at pH 4.2 from 4.0 microM to 0.5 microM. Residual 6S activity present in the skin fibroblast homogenates from MPS IIID patients was characterized for activity towards GlcNAc6S-IdoA2S-anM6S and observed to have similar pH optima and Km values to normal skin fibroblast 6S activities, although the residual 6S activity was less than 1% of the normal control range.

Chromatography, Gel↗
PubMed1993

Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides.

Glucosamine-6-phosphate deaminase is an oligomeric protein composed of six identical 29.7 kDa subunits. Each subunit has four cysteine residues located at positions 118, 219, 228 and 239. We have previously shown that Cys-118 and Cys-239 form a pair of vicinal thiols, the reactivity of which changes with the allosteric transition. The site-directed mutations Cys-->Ser corresponding to the other two cysteine residues have been constructed, as well as some selected multiple mutations involving the four cysteines. Thiol and disulphide measurements on the wild-type and mutant enzymes indicate that thiols from Cys-219 are oxidized and form interchain disulphide bonds. The disulphide-linked dimer was demonstrated by SDS/PAGE. This result is consistent with preliminary crystallographic data and thermal denaturation studies, and strongly suggests that glucosamine-6-phosphate deaminase is a trimer of disulphide-linked dimers. The mutant forms of the deaminase lacking the interchain disulphide bond or the thiol at Cys-228 are both stable hexamers showing the same sensitivity to urea denaturation as the wild-type protein. Furthermore, these Cys-->Ser mutants display the same kinetics and allosteric properties as those already described for the wild-type enzyme.

Aldose-Ketose Isomerases↗
PubMed2003

Selective activation of N-acyl-D-glucosamine 2-epimerase expression in failing human heart ventricular myocytes.

BACKGROUND: O-linked N-acyl-glycosylation may regulate protein function by competing with phosphorylation of serine residues. Availability of substrate for this process is regulated, in part, by N-Acyl-D-glucosamine 2-epimerase (NAGE), which interconverts N-acetyl-glucosamine (GlcNAc) and N-acetylmannosamine (ManNAc). NAGE is also a putative renin-binding protein. This study tested the hypothesis that NAGE is present in the human heart and that NAGE expression is increased in the failing human heart. METHODS AND RESULTS: Ribonuclease protection assays (RPAs) demonstrated increased NAGE gene expression in failing hearts from subjects with idiopathic dilated and ischemic cardiomyopathies compared with nonfailing hearts. In situ reverse transcriptase-polymerase chain reaction, using primers designed to localize NAGE mRNA, demonstrated that, in nonfailing hearts, NAGE gene expression was restricted to endothelial cells and not detectable in cardiac myocytes. However, in failing human hearts NAGE gene expression was selectively activated in cardiac myocytes, but not endothelial cells. Immunohistochemistry confirmed that the pattern of NAGE protein expression corresponded to the pattern of gene expression. CONCLUSIONS: NAGE gene and protein expression were selectively activated in left ventricular myocytes from end-stage failing human hearts.

Adolescent↗
PubMed1971

Kinetics of insulin release from the perfused rat pancreas caused by glucose, glucosamine, and galactose.

Under appropriate conditions, not only glucose but also glucosamine and galactose can serve as potent stimulants for insulin release from the isolated, perfused rat pancreas. Since galactose and, probably, glucosamine are not metabolized in the islets, and since these three compounds have in all likelihood common sites of action, it is postulated that a glucoreceptor of broad specificity is involved in the mechanism of insulin release, and that metabolism of glucose is not an essential part of the releasing action of this sugar.

Adenylyl Cyclases↗
PubMed1998

A family of human beta3-galactosyltransferases. Characterization of four members of a UDP-galactose:beta-N-acetyl-glucosamine/beta-nacetyl-galactosamine beta-1,3-galactosyltransferase family.

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:beta-N-acetyl-glucosamine beta-1, 3-galactosyltransferase, designated beta3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to beta3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP-galactose:beta-N-acetyl-glucosamine beta-1, 3-galactosyltransferase (beta3Gal-T2) with similar kinetic properties as beta3Gal-T1. Another gene represented a UDP-galactose:beta-N-acetyl-galactosamine beta-1, 3-galactosyltransferase (beta3Gal-T4) involved in GM1/GD1 ganglioside synthesis, and this gene was highly similar to a recently reported rat GD1 synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. beta3Gal-T1 mRNA was expressed in brain, beta3Gal-T2 was expressed in brain and heart, and beta3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. beta3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous beta3-galactosyltransferase genes.

Amino Acid Sequence↗
PubMed2004

Biochemical characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-dehydrogenase involved in O-antigen biosynthesis in Pseudomonas aeruginosa PAO1.

WbpA (PA3159) is an enzyme involved in the biosynthesis of unusual di-N-acetyl-d-mannosaminuronic acid-derived sugar nucleotides found in the O antigen of Pseudomonas aeruginosa PAO1 (serotype O5). The wbpA gene that encodes this enzyme was cloned into pET-28a, overexpressed as a histidine-tagged fusion protein, and purified by nickel chelation chromatography. Capillary electrophoresis was used to examine substrate conversion by WbpA, and the data revealed that WbpA is a UDP-N-acetyl-D-glucosamine 6-dehydrogenase (EC 1.1.1.136), which uses NAD(+) as a coenzyme. The enzyme reaction product was purified by HPLC and analyzed using NMR spectroscopy. Our results showed unequivocally that the product of the WbpA reaction is UDP-N-acetyl-d-glucosaminuronic acid. WbpA requires either NH(4)(+) or K(+) for activity and the accompanying anions exert secondary effects on activity consistent with their ranking in the Hofmeister series. Kinetic analysis showed positive cooperativity with respect to UDP-N-acetyl-d-glucosamine binding with a K(0.5) of 94 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.8. In addition, WbpA has a K(0.5) for NAD(+) of 220 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.1. The oligomerization state of WbpA was analyzed by gel filtration, dynamic light scattering, and analytical ultracentrifugation, with all three techniques indicating that WbpA exists as a trimer in solution. However, tertiary structure predictions suggested a tetramer, which was supported by data from transmission electron microscopy. The electron micrograph of negatively stained WbpA samples revealed structures with 4-fold symmetry.

Base Sequence↗
PubMed2005

Anticandidal properties of N-acylpeptides containing an inhibitor of glucosamine-6-phosphate synthase.

A series of N-acyl peptides 1-9, containing an inhibitor of glucosamine-6-phosphate synthase have been synthesised and tested against Candida strains. N-Acylated peptides inhibit glucosamine-6-phosphate synthase in cell free extracts from Candida albicans. Antifungal activities of the tested compounds correlated with their lipophilic properties. Peptides acylated with decanoic acid were found to be the most potent in the series. N-decanoylpeptides also showed activity against Candida albicans Gu5 resistant mutant with Cdr1 and Cdr2 drug extrusion proteins that causes MDR by an active efflux mechanism.

Antifungal Agents↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of N-acyl-D-glucosamine 2-epimerase from porcine kidney.

N-Acyl-D-glucosamine 2-epimerase from porcine kidney, which was cloned and expressed in Escherichia coli, was crystallized by the vapor-diffusion method, using polyethylene glycol and ammonium acetate as precipitants. The crystals were resistant to X-ray radiation damage and diffracted to more than 2.0 A resolution. The diffraction pattern indicated that the crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell dimensions of a = 78.1, b = 97.2, and c = 100.7 A. It is supposed that the asymmetric unit consists of two N-acyl-D-glucosamine 2-epimerase molecules. Collection of data on the native crystals indicated that they are suitable for X-ray structural analysis.

Animals↗
PubMed1984

D-glucosaminate dehydratase: spectrometric properties of the enzyme-bound pyridoxal 5'-phosphate.

The holoenzyme of D-glucosaminate dehydratase [EC 4.2.1.26] from Agrobacterium radiobacter showed absorption peaks at 280 and 415 nm with a shoulder in the region of 320 to 330 nm. The treatment of the enzyme with hydroxylamine followed by dialysis led to disappearance of both the absorption peak at 415 nm and the shoulder, giving the apoenzyme. The fluorescence excitation maximum of the holoenzyme was at 320 nm with a shoulder at 420 nm (emission at 510 nm), and the emission maxima were at 420 nm with a shoulder at 370 nm (excitation at 320 nm) and at 510 nm (excitation at 420 nm). The holoenzyme showed a negative circular dichroic band at 418 nm and a positive shoulder at around 320 nm. Reduction of the holoenzyme with sodium borohydride caused a loss of the absorption peak at 415 nm with a concomitant increase of 325 nm absorbance and an irreversible loss of the activity. The occurrence of epsilon-N-pyridoxyllysine in the acid hydrolysate of the reduced enzyme showed that D-glucosaminate dehydratase contains a catalytically essential lysine residue whose epsilon-amino group binds the 4-formyl group of pyridoxal 5'-phosphate to form a Schiff base. The plots of absorption of the apoenzyme against the amount of pyridoxal 5'-phosphate added showed that four and two molar equivalents of the cofactor bind to the apoenzyme and subunit, respectively. The biphasic nature of the spectrometric titration curve of the apoenzyme with pyridoxal 5'-phosphate and the two Km values obtained for the cofactor suggest the occurrence of two distinct types of binding sites for pyridoxal 5'-phosphate in the enzyme.

Circular Dichroism↗
PubMed1995

Glucosamine 6-phosphate deaminase in Plasmodium falciparum.

The pathways of glucose utilization for energy production in the malaria parasite, Plasmodium falciparum, have been studied extensively. Little is known, however, about the reactions by which glucose is converted into complex carbohydrates in the parasite, and knowledge of the catabolism of these substances is likewise scanty. The present investigation was undertaken to determine whether the parasites possess a key enzyme of glucosamine catabolism, i.e. glucosamine 6-phosphate deaminase (EC 5.3.1.40), which catalyses the conversion of the sugar phosphate to fructose 6-phosphate and ammonia. Lysates of Plasmodium-infected erythrocytes had substantially higher deaminase activity than control samples from normal erythrocytes, and an even higher specific activity was observed in extracts of isolated parasites, amounting to 20-40 times that of uninfected cells. Anion exchange chromatography indicated that the parasite deaminase eluted in a retarded position when compared to the elution profile of the erythrocyte enzyme. The charge difference suggested by these findings was established more directly by chromatofocusing, which indicated pI values of 6.85 and 8.55 for the parasite and erythrocyte deaminases, respectively. Other differences were also observed, notably a greater thermolability on the part of the parasite enzyme. These results indicated that the parasites synthesize a specific deaminase that is distinct from the normal erythrocyte enzyme. Studies on synchronized parasite cultures further indicated that the parasite deaminase is developmentally regulated, because a dramatic increase in activity levels occurred during the later stages of parasite development.

Aldose-Ketose Isomerases↗
PubMed1991

Possible involvement of Lys603 from Escherichia coli glucosamine-6-phosphate synthase in the binding of its substrate fructose 6-phosphate.

Pyridoxal 5'-phosphate is a competitive inhibitor of glucosamine-6-phosphate synthase with respect to the substrate fructose 6-phosphate. Irreversible inactivation of pyridoxal-5'-phosphate-treated enzyme with [14C]-cyanide resulted in covalent incorporation of close to 1 mol pyridoxal 5'-phosphate/mol enzyme subunit. The enzyme-pyridoxal-5'-phosphate complex could also be inactivated by reduction with NaBH3CN. Sequence analysis of the unique radioactively labelled tryptic peptide, resulting from inactivation with [3H]NaBH3CN, identified the C-terminal nonapeptide encompassing the modified Lys603. The presence of fructose 6-phosphate protected this residue from pyridoxylation. Direct evidence that a lysine residue is involved in the binding of the substrate as a Schiff base came from the isolation at 4 degrees C of a enzyme-fructose-6-phosphate complex in a 1:1 molar ratio. Treatment of the enzyme-[14C]fructose-6-phosphate complex with NaBH3CN revealed one site of modification in the tryptic peptide map. In contrast, trapping the same complex with potassium cyanide resulted in the isolation of several radiolabelled peptides containing lysines which could potentially bind fructose 6-phosphate. However, since the radioactivity was not specifically associated with the lysine residues, it is suggested that these 14C-labelled peptides resulted from the decomposition of an unstable alpha,alpha'-dihydroxyaminonitrile adduct rather than from a lack of specificity of fructose 6-phosphate fixation. Lys603 is then the candidate of choice for fructose 6-phosphate binding since it lies at or near the active site as demonstrated by the trapping experiments with pyridoxal 5'-phosphate described above, and among the lysines which belong to the sugar-binding domain this is the only one conserved between the three members of the purF, glutamine-dependent, amidotransferase subfamily which include the glucosamine-6-phosphate synthase from Escherichia coli, Saccharomyces cerevisiae and the Rhizobium nodulation protein NodM.

Amino Acid Sequence↗
PubMed1997

Affinity labeling of Escherichia coli glucosamine-6-phosphate synthase with a fructose 6-phosphate analog--evidence for proximity between the N-terminal cysteine and the fructose-6-phosphate-binding site.

Glucosamine-6-phosphate synthase (GlcNP-synthase) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using the gamma-amide functionality of glutamine as the nitrogen source. In the absence of glutamine, GlcNP-synthase was recently found to catalyze the formation of glucose 6-phosphate corresponding to a phosphoglucoisomerase-like activity. Here we report active-site directed, irreversible inhibition of Escherichia coli GlcNP-synthase (k(inact) = 0.60 +/- 0.05 min(-1), Kirr = 1.40 +/- 0.20 mM) by anhydro-1,2-hexitol 6-phosphates previously known as irreversible inhibitors of phosphoglucoisomerase. Enzyme inactivation with the tritiated affinity label, followed by tryptic digestion and purification of the radioactive fragments, allowed identification of three peptides. Two of them, accounting for 54% of the recovered radioactivity, are believed to result from the nucleophilic attack of side-chain carboxylates of Glu255 and Glu258 and thiol of Cys300 of the fructose-6-phosphate-binding site on the epoxide functionality of the inhibitor. The major peptide corresponds to derivatization of the N-terminal cysteine from the glutamine-binding site by the inhibitor. These results provide evidence for the close proximity of glutamine and fructose-6-phosphate-binding sites recently suggested by Bearne [Bearne, S. L. (1996) J. Biol. Chem. 271, 3052-3057].

Affinity Labels↗
PubMedDate not supplied

Giardia intestinalis glucosamine 6-phosphate isomerase: the key enzyme to encystment appears to be controlled by ubiquitin attachment.

The cyst wall of the parasitic protozoan, Giardia intestinalis, is composed of a polymer of N-acetylgalactosamine, the precursor of which is synthesized by an inducible enzyme pathway. The first enzyme in this pathway, glucosamine 6-phosphate isomerase, is transcriptionally regulated. During encystment and in mature cysts this isomerase appears to be modified by ubiquitin attachment. Thus, it might be targeted for destruction by an ubiquitin-mediated pathway, suggesting that glucosamine 6-phosphate isomerase expression is tightly regulated.

Aldose-Ketose Isomerases↗
PubMed2002

N-acetyl-D-glucosamine specific hemagglutinin receptor of Vibrio cholerae O1 in chicken erythrocyte membranes.

N-Acetyl-D-glucosamine specific cell-associated hemagglutinin (HA)/lectin, previously purified from a strain of Vibrio cholerae O1, had been established as an adhesin molecule of V. cholerae O1 cells. This communication records the isolation and purification of the glycoprotein receptor of the N-acetyl-D-glucosamine specific HA of the V. cholerae O1 strain from chicken erythrocyte membranes. The most salient feature of this study is that the pretreatment of partially purified glycoprotein with purified HA could completely inhibit the hemagglutinating activity of the V. cholerae O1 strain with chicken erythrocytes.

Acetylglucosamine↗