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PubMed2005

Construction, purification, and functional characterization of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli.

Expression plasmids containing recombinant genes encoding three His(6)-tagged versions of the enzyme, glucosamine-6-phosphate synthase from Candida albicans, were constructed and overexpressed in Escherichia coli. The gene products were purified by metal-affinity chromatography to near homogeneity with 77-80% yield and characterized in terms of size and enzymatic properties. Presence of oligohistidyl tags at either of two ends did not affect enzyme quarternary structure but strongly influenced its catalytic activity. The His6-N-tagged enzyme completely lost an ability of glucosamine-6-phosphate formation and amidohydrolase activity but retained the hexosephosphate-isomerising activity. On the other hand, two His6-C-tagged versions of glucosamine-6-phosphate synthase exhibited amidohydrolase activity almost equal to that of the wild-type enzyme but only 18% of its hexosephosphate-isomerising activity and about 1.5% of the synthetic activity.

Candida albicans↗

PubMed1988

Human glucosamine-6-sulfatase cDNA reveals homology with steroid sulfatase.

Glucosamine-6-sulfatase is a lysosomal enzyme which degrades glycosaminoglycans and is deficient in mucopolysaccharidosis type IIID. Human liver contains two major active forms of glucosamine-6-sulfatase, form A which has a single 78 kDa polypeptide and form B which has two polypeptides of 48 kDa and 32 kDa. A 1761 base pair cDNA clone encoding the complete 48 kDa polypeptide of form B was isolated. Form A is shown to be processed to form B with the 48 kDa polypeptide C-terminal to the 32 kDa polypeptide, and it is shown that C-terminal processing is limited to a region of thirty amino acids. The glucosamine-6-sulfatase sequence reveals homology with steroid sulfatase, a microsomal enzyme.

Amino Acid Sequence↗

PubMed1998

Involvement of the C terminus in intramolecular nitrogen channeling in glucosamine 6-phosphate synthase: evidence from a 1.6 A crystal structure of the isomerase domain.

BACKGROUND: Glucosamine 6-phosphate synthase (GlmS) catalyses the first step in hexosamine metabolism, converting fructose-6P (6 phosphate) into glucosamine-6P using glutamine as a nitrogen source. GlmS is a bienzyme complex consisting of two domains that catalyse glutamine hydrolysis and sugar-phosphate isomerisation, respectively. Knowledge of the three-dimensional structure of GlmS is essential for understanding the general principles of catalysis by ketol isomerases and the mechanism of nitrogen transfer in glutamine amidotransferases. RESULTS: The crystal structure of the isomerase domain of the Escherichia coli GlmS with the reaction product, glucosamine-6P, has been determined at 1.57 A resolution. It is comprised of two topologically identical subdomains, each of which is dominated by a nucleotide-binding motif of a flavodoxin type. The catalytic site is assembled by dimerisation of the protein. CONCLUSIONS: The isomerase active site of GlmS seems to be the result of evolution through gene duplication and subsequent dimerisation. Isomerisation of fructose-6P is likely to involve the formation of a Schiff base with Lys603 of the enzyme, the ring-opening step catalysed by His504, and the proton transfer from C1 to C2 of the substrate effected by Glu488. The highly conserved C-terminal fragment of the chain may play a key role in substrate binding, catalysis and communication with the glutaminase domain. The corresponding sequence pattern DXPXXLAK[SC]VT (in single-letter amino-acid code, where X is any amino acid and letters in brackets indicate that either serine or cysteine may take this position) may be considered as a fingerprint of GlmS.

Amino Acid Sequence↗

PubMed1995

Asymmetric allosteric activation of Escherichia coli glucosamine-6-phosphate deaminase produced by replacements of Tyr 121.

Tyrosine 121, a residue located in a alpha-helical polypeptide segment of glucosamine 6-phosphate deaminase from Escherichia coli, has recently been proposed to have a role in the binding of the allosteric activator N-acetyl-D-glucosamine 6-phosphate. Accordingly, the site-directed mutants Tyr 121-Thr and Tyr 121-Trp were constructed, to assess experimentally the role of Tyr 121 in the allosteric function of the enzyme. The kinetic study of both mutant forms revealed that the replacements caused striking changes in allosteric activator binding and allosteric properties, when compared to the wild-type enzyme. While the wild-type deaminase behaves as a classical allosteric K-system which can be described by the allosteric concerted model, both mutant forms present an asymmetric behavior toward the allosteric activator, which can be described as two distinct half-of-the-sites allosteric activation steps occurring with different affinities for the N-acetyl-D-glucosamine 6-phosphate. During the first (high affinity) activation phase, the mutant forms of deaminase behave as mixed K/V allosteric enzyme. The biphasic activation curve was also demonstrated by direct binding measurements of the 14C-labeled activator to Tyr 121-Trp and Tyr 121-Thr deaminases. The kinetic analysis of these mutant forms also showed that the threonine replacement produced an important distortion of the enzyme structure reflected in a considerable decrease of its catalytic efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldose-Ketose Isomerases↗

PubMed1992

Identification of two cysteine residues forming a pair of vicinal thiols in glucosamine-6-phosphate deaminase from Escherichia coli and a study of their functional role by site-directed mutagenesis.

The nucleotide sequence of the nagB gene in Escherichia coli, encoding glucosamine-6-phosphate deaminase, located four cysteinyl residues at positions 118, 219, 228, and 239. Chemical modification studies performed with the purified enzyme had shown that the sulfhydryl groups of two of these residues form a vicinal pair in the enzyme and are easily modified by thiol reagents. The allosteric transition to the more active conformer (R), produced by the binding of homotropic (D-glucosamine 6-phosphate or 2-deoxy-2-amino-D-glucitol 6-phosphate) or heterotropic (N-acetyl-D-glucosamine 6-phosphate) ligands, completely protected these thiols against chemical modification. Selective cyanylation of the vicinal thiols with 2-nitro-5-(thiocyanato)benzoate, followed by alkaline hydrolysis to produce chain cleavage at the modified cysteines, gave a pattern of polypeptides which allowed us to identify Cys118 and Cys239 as the residues forming the thiol pair. Subsequently, three mutated forms of the gene were constructed by oligonucleotide-directed mutagenesis, in which one or both of the cysteine codons were changed to serine. The mutant proteins were overexpressed and purified, and their kinetics were studied. The dithiol formed by Cys118 and Cys239 was necessary for maximum catalytic activity. The single replacements and the double mutation affected catalytic efficiency in a similar way, which was also identical to the effect of the chemical block of the thiol pair. However, only one of these cysteinyl residues, Cys239, had a significant role in the allosteric transition, and its substitution for serine reduced the allosteric interaction energy, due to a lower value of KT.

Aldose-Ketose Isomerases↗

PubMed1987

Glucosamine synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location.

L-Glutamine:D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction, as reported for more impure preparations [Gosh, S., Blumenthal, H. J., Davidson, E., & Roseman, S. (1960) J. Biol. Chem. 235, 1265]. The enzyme has a Km of 2 mM for fructose 6-phosphate, a Km of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence [Walker, J. E., Gay, J., Saraste, M., & Eberle, N. (1984) Biochem. J. 224, 799]. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inactivated by the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation.

Amino Acids↗

PubMed1980

The isolation and preliminary characterization of N-acetyl-D-glucosamine kinase from rat kidney and liver.

1. Procedures for the extensive purification in high yield of N-acetyl-D-glucosamine kinase from rat liver and kidney are described. The separation of this enzyme from hepatic glucokinase depended primarily on their differing behaviour on an affinity column of Sepharose--N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose. 2. This N-acetyl-D-glucosamine kinase also catalyses the phosphorylation of N-acetyl-D-mannosamine and, at a lower rate, several other sugar analogues, including D-glucose. 3. A comparison of the behaviour of the enzyme during gel filtration and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels suggests that N-acetyl-D-glucosamine kinase is a symmetrical dimer of mol.wt. 80000.

Acetylglucosamine↗

PubMed2003

Synthesis of paucimannose N-glycans by Caenorhabditis elegans requires prior actions of UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I, alpha3,6-mannosidase II and a specific membrane-bound beta-N-acetylglucosaminidase.

We have previously reported three Caenorhabditis elegans genes ( gly-12, gly-13 and gly-14 ) encoding UDP- N -acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2- N -acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid and complex N-glycan synthesis. GLY-13 was shown to be the major GnT I in worms and to be the only GnT I cloned to date which can act on [Manalpha1,6(Manalpha1,3)Manalpha1,6](Manalpha1,3)Manbeta1, 4GlcNAcbeta1,4GlcNAc-R, but not on Manalpha1,6(Manalpha1,3)Manbeta1- O -R substrates. We now report the kinetic constants, bivalent-metal-ion requirements, and optimal pH, temperature and Mn(2+) concentration for this unusual enzyme. C. elegans glycoproteins are rich in oligomannose (Man(6-9)GlcNAc(2)) and 'paucimannose' Man(3-5)GlcNAc(2)(+/-Fuc) N-glycans, but contain only small amounts of complex and hybrid N-glycans. We show that the synthesis of paucimannose Man(3)GlcNAc(2) requires the prior actions of GnT I, alpha3,6-mannosidase II and a membrane-bound beta- N -acetylglucosaminidase similar to an enzyme previously reported in insects. The beta- N -acetylglucosaminidase removes terminal N -acetyl-D-glucosamine from the GlcNAcbeta1, 2Manalpha1,3Manbeta- arm of Manalpha1,6(GlcNAcbeta1,2Manalpha1,3) Manbeta1,4GlcNAcbeta1,4GlcNAc-R to produce paucimannose Man(3)GlcNAc(2) N-glycan. N -acetyl-D-glucosamine removal was inhibited by two N -acetylglucosaminidase inhibitors. Terminal GlcNAc was not released from [Manalpha1,6(Manalpha1,3)Manalpha 1,6] (GlcNAcbeta1,2Manalpha1,3)Manbeta1,4GlcNAcbeta1,4GlcNAc-R nor from the GlcNAcbeta1,2Manalpha1,6Manbeta- arm. These findings indicate that GLY-13 plays an important role in the synthesis of N-glycans by C. elegans and that therefore the worm should prove to be a suitable model for the study of the role of GnT I in nematode development.

Acetylglucosaminidase↗

PubMed2002

Glucosamine:fructose-6-phosphate aminotransferase: gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegypti.

Glucosamine:fructose-6-phosphate aminotransferase (GFAT) catalyses the formation of glucosamine 6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. The final product of the hexosamine pathway, UDP-N-acetyl glucosamine, is an active precursor of numerous macromolecules containing amino sugars, including chitin in fungi and arthropods. Chitin is one of the essential components of insect cuticle and peritrophic matrix. The peritrophic matrix is produced in the midgut of mosquitoes in response to bloodfeeding, and may affect vector competence by serving as a physical barrier to pathogens. It is hypothesized that GFAT plays a regulatory role in biosynthesis of chitin and peritrophic matrix formation in insects. We cloned and sequenced the GFAT gene (AeGfat-1) and its 5' regulatory region from Aedes aegypti. There is no intron in AeGfat-1 and there are two potential transcription start sites. AeGfat-1 cDNA is 3.4 kb in length and its putative translation product is 75.4 kDa. The amino acid sequence of GFAT is highly conserved in lower and higher eukaryotes, as well as in bacteria. AeGfat-1 message is constitutively expressed but is gradually up-regulated in the midgut after bloodfeeding. The putative regulatory region of the gene contains the ecdysone response element, E74, and Broad complex motifs, similar to what is found in the glutamine synthetase gene in Ae. aegypti. Results suggest that Ae. aegypti GFAT-1 may have a regulatory role in chitin biosynthesis and peritrophic matrix formation, and probably is under the regulation of ecdysteroids.

5' Untranslated Regions↗

PubMed1972

Synthesis and secretion of gammaglobulin by lymph node cells: the acquisition of carbohydrate residues of immunoglobulin in relation to interchain disulfide bond formation (heavy and light chains-murine myeloma-mannose-glucosamine-galactose).

Acquisition of carbohydrates in the disulfide-linked heavy (H) and light (L) chain molecules of murine myeloma (ADJPC5), i.e., HH, HHL, and LHHL, was investigated. That some mannose and glucosamine residues are acquired by immunoglobulin precursor molecules was demonstrated by the detection of glucosamine and mannose in HH, HHL, and LHHL. In contrast, galactose was observed solely in LHHL molecules, which have an identical electrophoretic mobility to the secreted product. Furthermore, as judged from cells incubated with [(3)H]leucine, the more juvenile molecules HH and HHL were predominant in the rough microsome fraction, whereas LHHL was the principal molecular species in the smooth microsome fraction. Findings of this type were not observed in rabbit lymph node cells. Thus, galactose, as well as mannose and glucosamine, were found in the more juvenile molecule known for this species (HL). Moreover, the ratio of HL:LHHL, as judged from cells incubated with [(3)H]leucine, was about the same in rough and smooth microsomes.

Animals↗

PubMed2005

Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase.

Glucosamine-6P synthase catalyzes the synthesis of glucosamine-6P from fructose-6P and glutamine and uses a channel to transfer ammonia from its glutaminase to its synthase active site. X-ray structures of glucosamine-6P synthase have been determined at 2.05 Angstroms resolution in the presence of fructose-6P and at 2.35 Angstroms resolution in the presence of fructose-6P and 6-diazo-5-oxo-L-norleucine, a glutamine affinity analog that covalently modifies the N-terminal catalytic cysteine, therefore mimicking the gamma-glutamyl-thioester intermediate formed during hydrolysis of glutamine. The fixation of the glutamine analog activates the enzyme through several major structural changes: 1) the closure of a loop to shield the glutaminase site accompanied by significant domain hinging, 2) the activation of catalytic residues involved in glutamine hydrolysis, i.e. the alpha-amino group of Cys-1 and Asn-98 that is positioned to form the oxyanion hole, and 3) a 75 degrees rotation of the Trp-74 indole group that opens the ammonia channel.

Ammonia↗

PubMed1998

Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity.

Glucosamine-6-phosphate deaminase (GNPDA) catalyzes the conversion of glucosamine-6-phosphate to fructose-6-phosphate, a reaction that under physiological conditions proceeds to the formation of fructose-6-phosphate. Though first identified in mammalian tissues in 1956, the enzyme has not previously been molecularly characterized in mammalian tissues, although a bacterial GNPDA has been cloned. Recently, a protein displaying similarity to bacterial GNPDA was purified and cloned from sperm extract. It was proposed that this protein was the factor, found in sperm extracts, that causes calcium oscillations in cells; thus, the protein was named 'oscillin.' We demonstrate that oscillin is the mammalian form of glucosamine 6-phosphate deaminase by showing that cloned oscillin has a robust GNPDA activity and can account for all such activity in mammalian tissues extracts. In situ hybridization and immunohistochemistry localize GNPDA selectively to tissues with high energy requirements such as the apical zone of transporting epithelia in the proximal convoluted tubules of the kidney and the small intestine; to neurons (but not glia) and especially to nerve terminals in the brain; and to motile sperm. Recombinant GNPDA and GNPDA purified to homogeneity from hamster sperm fail to elevate intracellular calcium when injected into mouse eggs over a wide range of concentrations under conditions in which sperm extracts elicit pronounced calcium oscillations. Thus, the calcium-releasing or oscillin activity of sperm extracts is due to a substance other than GNPDA. Since GNPDA is the sole enzyme linking hexosamine systems with glycolytic pathways, we propose that it provides a source of energy in the form of phosphosugar derived from the catabolism of hexosamines found in glycoproteins, glycolipids, and sialic acid-containing macromolecules. Evidence that GNPDA can regulate hexosamine stores comes from our observation that transfection of GNPDA into HEK-293 cells reduces cellular levels of sialic acid.

Aldose-Ketose Isomerases↗

PubMed2004

Isolation, purification, and characterization of poly-N-acetyl glucosamine use as a hemostatic agent.

BACKGROUND: A new polymeric material, poly-N-acetyl glucosamine (p-GlcNAc) fiber, has been identified and is effective in achieving hemostasis in surgical procedures and trauma. The p-GlcNAc material is purified from large-scale cultures of a marine microalga. METHODS: Poly-N-acetyl glucosamine materials have been formulated as films, sponges, gels, and microspheres. The polymer's structure has been characterized by chemical composition, carbohydrate analysis, spectroscopic techniques, intrinsic viscosity, and electron microscopy. RESULTS: Carbohydrate analyses indicate that the primary sugar present in p-GlcNAc is N-acetyl glucosamine. Elemental analyses yield percentage values for carbon, nitrogen, and hydrogen that support that the polymer is fully acetylated. Molecular weight determinations indicate that the polymer has a molecular weight of 2.0 x 10(6) Da. Fourier transform infrared, nuclear magnetic resonance, and circular dichroism spectral data have defined a unique tertiary structure. Biologic testing demonstrated that p-GlcNAc materials are fully biocompatible. CONCLUSION: The p-GlcNAc fiber has a unique beta-tertiary structure.

Acetylglucosamine↗

PubMed2004

Comparison of poly-N-acetyl glucosamine with commercially available topical hemostats for achieving hemostasis in coagulopathic models of splenic hemorrhage.

BACKGROUND: The hemostatic quality of the poly-N-acetyl glucosamine (p-GlcNAc) patch was compared with a fibrin sealant, fibrin bandage, and cellulose patch. METHODS: A 2 x 2-cm capsular strip to a depth of 3 mm of the swine spleen was used as a source of bleeding. Splenic lacerations were created in hemophilia B dogs and treated with p-GlcNAc and Surgicel. Wounds were created in rabbits and treated with p-GlcNAc at 37degreesC and after keeping body core temperature at 29degreesC. RESULTS: Poly-N-acetyl glucosamine was able to achieve hemostasis with greater efficacy than either of the fibrin-based bandages. In the hemophilia B dog study, p-GlcNAc significantly outperformed Surgicel, with p-GlcNAc achieving hemostasis in 75% of the treated wounds compared with 17% for the cellulose patch. The hypothermia study demonstrated that p-GlcNAc is equally effective at 29degreesC and at 37degreesC. CONCLUSION: Poly-N-acetyl glucosamine was effective at controlling bleeding in animals with experimentally induced or genetic coagulopathic disorders.

Acetylglucosamine↗

PubMed1972

Location of glycoproteins that contain glucosamine in plant tissues.

When radioactive d-glucosamine is provided to Acer pseudoplatanus cells in liquid culture in order to label those glycoproteins that contain amino sugars, it is incorporated predominantly into a crude cell wall fraction. This observation was confirmed histologically by preparing autoradiographs of thin tissue sections from plasmolyzed cells. Highly purified cell wall material from unlabeled cells has also been shown to contain small amounts of glucosamine. Similarly, about one-half of the amino sugar recovered from cultured cells of Nicotiana tabacum is present in their cell walls. In corn roots, however, the labeled glycoproteins that are formed after glucosamine incorporation are predominantly cytoplasmic and not deposited outside the protoplast.

Journal Article↗

PubMed1975

The Incorporation of d-Glucosamine into Glycolipids and Glycoproteins of Membrane Preparations from Phaseolus aureus Hypocotyls.

Radioactivity from d-glucosamine-(14)C is incorporated into particulate fractions of hypocotyls of Phaseolus aureus (mung bean) seedlings. Polyacrylamide gel electrophoresis of the sodium dodecyl sulfate-solubilized materials revealed that several polypeptide components varying considerably in molecular weight had become radioactive during the incubation. A considerable amount of (14)C was also recovered in lipid. Equilibrium centrifugation of the particulate material, isolated by initial centrifugation at 100,000 times gravity on sucrose density gradients revealed that radioactivity was recoverable in all of the membrane fractions along the gradient. It is suggested that glycoproteins and glycolipids containing amino sugar are normal constituents of such membranes. The ability of the particulate preparations to catalyze the transfer of N-acetyl-d-glucosamine from uridine diphospho-N-acetyl-d-glucosamine to endogenous acceptor material was also tested. Transfer was optimal at around pH 9 and in the presence of 10 mm Mg(2+), and it occurred largely into an unidentified lipid fraction. After equilibrium centrifugation of crude membrane material on sucrose gradients, a number of distinct fractions could be detected which would catalyze the transfer reaction. Uridine diphospho-d-glucose transferase activity showed a similar but not identical distribution along the gradient.

Journal Article↗

PubMed1980

Incorporation of [C]Glucosamine and [C]Mannose into Glycolipids and Glycoproteins in Cotyledons of Pisum sativum L.

Developing pea cotyledons incorporate radioactivity in vivo from [(14)C]glucosamine and [(14)C]mannose into glycolipids and glycoproteins. Several different lipid components are labeled including neutral, ionicnonacidic, and acidic lipids. The acidic lipids labeled in vivo appear similar to the polyisoprenoid lipid intermediates formed in vitro in pea cotyledons. Radioactivity from [(14)C]glucosamine and [(14)C]mannose is also incorporated into glycopeptides. Considerable redistribution of [(14)C]mannose into other glycosyl components found in endogenous glycoproteins is observed. An N-acetylglucosamine to asparagine glycopeptide linkage has been isolated from [(14)C]glucosamine-labeled glycoproteins.

Journal Article↗

PubMed2005

Crystallization and preliminary X-ray analysis of the isomerase domain of glucosamine-6-phosphate synthase from Candida albicans.

Glucosamine-6-phosphate synthase (EC 2.6.1.16) catalyses the first and practically irreversible step in the hexosamine metabolism pathway, the end product of which, uridine 5'-diphospho-N-acetyl D-glucosamine, is an essential substrate for assembly of the cell wall. The isomerase domain, consisting of residues 346-712 (42 kDa), of glucosamine-6-phosphate synthase from Candida albicans has been crystallized. X-ray analysis revealed that the crystals belonged to space group I4, with unit-cell parameters a = b = 149, c = 103 A. Diffraction data were collected to 3.8 A. Preliminary results from molecular replacement using the homologous bacterial monomer reveal that the asymmetric unit contains two monomers that resemble a bacterial dimer. The crystal lattice consists of pairs of such symmetry-related dimers forming elongated tetramers.

Aldose-Ketose Isomerases↗