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.     

An efficient method for production of uridine 5'-diphospho-N-acetylglucosamine

Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) has been synthesized by a yeast-based method from 5'-UMP and glucosamine, in which yeast cells catalyze the conversion of 5'-UMP to 5'-UTP and provide enzymes involved in UDP-GlcNAc synthesis using 5'-UTP and glucosamine as substrates. However, this conventional method is not suitable for practical production of UDP-GlcNAc because of the low yield of the product. We found that the yqgR gene product of Bacillus subtilis, which has been identified as a glucokinase, can catalyze the phosphorylation of N-acetylglucosamine (GlcNAc) to give GlcNAc-6-phosphate, an intermediate of UDP-GlcNAc biosynthesis. The addition of the yqgR gene product to the yeast-based reaction system enabled us to synthesize UDP-GlcNAc using GlcNAc in place of glucosamine. The addition of two enzymes, GlcNAc-phosphate mutase and UDP-GlcNAc pyrophosphorylase, increased the yield of UDP-GlcNAc. Using this novel method, UDP-GlcNAc was produced at an amount of 78 mM from 100 mM 5'-UMP and 100 mM GlcNAc.     

Uridine sugar nucleotides modified in their nucleoside moieties and their effect on lipid-linked saccharide formationin vitro

Uridine diphospho glucose (UDP-Glc) and uridine diphospho N-acetylglucosamine (UDP-GlcNAc), modified in the uridine moiety by either periodate oxidation of the ribose ring or substitution at position 5 of the uracil ring with fluorine, have been tested as potential inhibitors of glucosyl monophosphoryl dolichol (Glc-P-Dol) or N,N-diacetylchitobiosyl pyrophosphoryl dolichol [GlcNAc)2-PP-Dol) assembly in chick embryo cell membranes. The periodate oxidised sugar nucleotides inhibited glycosyl transfer from their respective natural counterparts by 50% at 230 micron periodate oxidised UDP-Glc and 70 micron periodate oxidised UDP-GlcNAc respectively. Inhibition in both cases was irreversible and addition of exogenous Dol-P stimulated only the residual non-inhibited reaction. Periodate oxidised UDP-GlcNAc preferentially inhibited the transfer of GlcNAc to GlcNac-PP-Dol. The sugar nucleotide containing 5-fluorouridine were, on the other hand, alternative substrates for Glc-P-Dol or (GlcNAc)2-PP-Dol synthesis. FUDP-Glc was a good substrate for Glc-P-Dol formation; having Km and Vmax values equal to those of UDP-Glc, whereas FUDP-GlcNAc was a less efficient substrate for the formation of (GlcNAc)2-PP-Dol; having Km and Vmax values one half and one third respectively of those of UDP-GlcNAc.     

Crystal structures of N-acetylglucosamine-phosphate mutase, a member of the alpha-D-phosphohexomutase superfamily, and its substrate and product complexes

N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthetic process of UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a UDP sugar that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Thus, a specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis. AGM1 catalyzes the conversion of N-acetylglucosamine 6-phosphate (GlcNAc-6-P) into N-acetylglucosamine 1-phosphate (GlcNAc-1-P). This enzyme is a member of the alpha-D-phosphohexomutase superfamily, which catalyzes the intramolecular phosphoryl transfer of sugar substrates. Here we report the crystal structures of AGM1 from Candida albicans for the first time, both in the apoform and in the complex forms with the substrate and the product, and discuss its catalytic mechanism. The structure of AGM1 consists of four domains, of which three domains have essentially the same fold. The overall structure is similar to those of phosphohexomutases; however, there are two additional beta-strands in domain 4, and a circular permutation occurs in domain 1. The catalytic cleft is formed by four loops from each domain. The N-acetyl group of the substrate is recognized by Val-370 and Asn-389 in domain 3, from which the substrate specificity arises. By comparing the substrate and product complexes, it is suggested that the substrate rotates about 180 degrees on the axis linking C-4 and the midpoint of the C-5-O-5 bond in the reaction.     

Efficient chemoenzymatic synthesis of uridine 5′-diphosphate N-acetylglucosamine and uridine 5′-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes

Uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5′-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51?g/L) and UDP-GlcNTFA (46.54?g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.     

Solubilization and characterization of the initial enzymes of the dolichol pathway from yeast

Preparation and purification of substrate amounts of radioactive as well as non-radioactive dolichyl diphosphate N-acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I-III). The test conditions are described in detail. All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35-fold and 70-fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+. The Km values for UDP-N-acetylglucosamine for enzyme I and II were 15 and 10 muM, respectively, and for GDP-mannose (enzyme III) 7 muM. The apparent Km values for the lipophilic acceptor was 180 muM for enzyme I (dolichyl phosphate), 40 muM for enzyme II (dolichyl diphosphate N-acetylglucosamine) and 17 muM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol X h-1 X mg protein-1. All reactions were inhibited by nucleoside diphosphates.     

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

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

UDP-N-Acetyl-α-D-glucosamine as acceptor substrate of β-1,4-galactosyltransferase. Enzymatic synthesis of UDP-N-acetyllactosamine

The capacity of UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAc) as an in vitro acceptor substrate for beta-1,4-galactosyltransferase (beta4GalT1, EC 2.4.1.38) from human and bovine milk and for recombinant human beta4GalT1, expressed in Saccharomyces cerevisiae, was evaluated. It turned out that each of the enzymes is capable to transfer Gal from UDP-alpha-D-galactose (UDP-Gal) to UDP-GlcNAc, affording Gal(beta1-4)GlcNAc(alpha1-UDP (UDP-LacNAc). Using beta4GalT1 from human milk, a preparative enzymatic synthesis of UDP-LacNAc was carried out, and the product was characterized by fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy. Studies with all three beta4GalTs in the presence of alpha-lactalbumin showed that the UDP-LacNAc synthesis is inhibited and that UDP-alpha-D-glucose is not an acceptor substrate. This is the first reported synthesis of a nucleotide-activated disaccharide, employing a Leloir glycosyltransferase with a nucleotide-activated monosaccharide as acceptor substrate. Interestingly, in these studies beta4GalT1 accepts an alpha-glycosidated GlcNAc derivative. The results imply that beta4GalT1 may be responsible for the biosynthesis of UDP-LacNAc, previously isolated from human milk.     

A UDP-HexNAc:polyprenol-P GalNAc-1-P transferase (WecP) representing a new subgroup of the enzyme family

The Aeromonas hydrophila AH-3 WecP represents a new class of UDP-HexNAc:polyprenol-P HexNAc-1-P transferases. These enzymes use a membrane-associated polyprenol phosphate acceptor (undecaprenyl phosphate [Und-P]) and a cytoplasmic UDP-d-N-acetylhexosamine sugar nucleotide as the donor substrate. Until now, all the WecA enzymes tested were able to transfer UDP-GlcNAc to the Und-P. In this study, we present in vitro and in vivo proofs that A. hydrophila AH-3 WecP transfers GalNAc to Und-P and is unable to transfer GlcNAc to the same enzyme substrate. The molecular topology of WecP is more similar to that of WbaP (UDP-Gal polyprenol-P transferase) than to that of WecA (UDP-GlcNAc polyprenol-P transferase). WecP is the first UDP-HexNAc:polyprenol-P GalNAc-1-P transferase described.     

Dolichyl sulphate and H-phosphonate: enzymatic reactions with activated sugars.

Two phosphate-modified analogues of dolichyl phosphate were evaluated as substrates or inhibitors of the reactions catalyzed by mammalian microsomal enzymes. Dolichyl H-phosphonate could serve as an efficient acceptor for mannosyl and glucosyl transfer. The reaction products were chromatographically different from those formed from dolichyl phosphate. Lower activity of the H-phosphonate was observed for the reaction of N-acetylglucosaminyl phosphate transfer from UDP-GlcNAc. Dolichyl sulphate was shown not to serve as a substrate for the transfer of mannosyl (from GDP-Man), glucosyl (from UDP-Glc) or N-acetylglucosaminyl phosphate (from UDP-GlcNAc) residues in the presence of rat liver microsomes. Weak inhibitory properties of this analogue were demonstrated.     

Molecular evolution and functional divergence of UDP-hexose 4-epimerases

UDP-glucose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) and/or the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) in sugar metabolism. GalEs belong to the short-chain dehydrogenase/reductase superfamily, use a conserved ‘transient keto intermediate’ mechanism and have variable substrate specificity. GalEs have been classified into three groups based on substrate specificity: group 1 prefers UDP-Glc/Gal, group 3 prefers UDP-GlcNAc/GalNAc, and group 2 has comparable activities for both types of the substrates. The phylogenetic relationship and structural basis for the specificities of GalEs revealed possible molecular evolution of UDP-hexose 4-epimerases in various organisms. Based on the recent advances in studies on GalEs and related enzymes, an updated view of their evolutional diversification is presented.     

Role of the carboxyl-terminal region in the activity of N-acetylglucosamine 6-o-sulfotransferase-1

N-Acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-6 position of non-reducing N-acetylglucosamine. N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) is the first cloned GlcNAc6ST and is involved in the synthesis of the L-selectin ligand. We noticed conserved C-terminal segments among GlcNAc6STs and produced mutant enzymes to reveal the functional significance. Mutant enzymes were transiently expressed as fusion proteins with protein A in COS-7 cells, and some of them were purified to homogeneity by IgG Sepharose column chromatography. Deletion of a C-terminal segment (amino acid numbers 479-483) resulted in a complete loss of the activity, when assayed using GlcNAcbeta1-6ManOMe as a substrate. Upon site-directed mutagenesis of the C-terminal region, three mutants, L477A, L478A and L483A, exhibited reduced activity. The K(M )values for GlcNAcbeta1-6ManOMe of L477A and L478A were 4 times higher than the K(M) of the wild-type enzyme, while that of L483A was unchanged. On the other hand the K(M )for PAPS of L483A was 3 times higher than that of the wild-type enzyme, while the values of L477A and L478A were unchanged. Furthermore, the L477A mutant acted on a core 3 structure (GlcNAcbeta1-3GalNAc-pNP), while the wild-type enzyme does not. These results demonstrate a role for leucine residues in the C-terminal region in the enzymatic activity.     

Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase

We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, suggesting that alloxan indeed was inhibiting (OGT). In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0-10 mM alloxan, and OGT activity was measured directly by quantitating UDP-[(3)H]-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions, OGT activity was completely inhibited by 1 mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT, and as such, is the first OGT inhibitor described.     

Characterization of UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1,6-N-acetylglucosaminyltransferase V from a human hepatoma cell line Hep3B.

UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1, 6-N-acetylglucosaminyltransferase V (GlcNAcT-V) has been purified from cell extracts of the human hepatoma cell line, Hep3B, with 8.7% recovery. The purified enzymes had molecular masses of about 67 and 65 kDa on denaturated and natural conditions, respectively. The values of pI was 5.9. The GlcNAcT-V, when resolved by SDS-PAGE, was positive for Schiff staining, suggesting that the enzyme is glycoprotein. When GlcN,GlcN-biant-PA and UDP-GlcNAc were used as substrates, the enzyme displayed a temperature optimum of around 50 degrees C and optimum an pH of 6.5. The enzyme was stable in response to incubation from pH 4.5 to pH 10.5 at 4 degrees C for 24 h. The presence of UDP-GlcNAc and GlcN,GlcN-bi-PA protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn2+ ion; however, it was inhibited by Fe3+. The enzyme activity was inhibited by another series of NDP-sugars including ADP-, CDP-, GDP-, and TDP-GlcNAc. Studies on the activity of the enzyme toward a variety of pyridylaminated sugars showed that the enzyme is most active toward biantennary (GlcN,GlcN-bi-PA) sugars. The enzymes had apparent Km values of 1.28 and 5.8 mM for GlcN,GlcN-bi-PA and UDP-GlcNAc, respectively. In order to isolate the GlcNAcT-V gene, PCR primers of GNN-1 and GNN-8 were designed and the amplified PCR product carrying the gene was cloned and sequenced. Nucleotide sequence analysis showed a 2220-bp open reading frame encoding a 740-amino-acid protein. This was almost same as the previously reported human sequences, except for some sequence differences in three amino acids. The three amino acid changes were as follows: 375V --> L, 555T --> R, and 592A --> G. These studies represent the detailed characterization of a purified GlcNAcT-V from human hepatoma cell Hep3B.     

Chitosome-like vesicles from gamma particles of Blastocladiella emersonii synthesize chitin

When gamma particles isolated from the aquatic fungus, Blastocladiella emersonii, were incubated in a supernatant derived from a homogenate of zoospores previously triggered to encyst with 50 mM KCl, they exhibited a three-fold increase in chitin synthetase activity and produced chitosome-like vesicles. Passage of such vesicles through a linear sucrose gradient resulted in a symmetrical distribution of chitin synthetase activity over a broad portion of the gradient, and the specific activity of the peak fraction was seven times greater than that of the gamma particles. After isopycnic sucrose gradient centrifugation, chitin synthetase activity occurred in a band of particles with a peak buoyant density of 1.18 g/cm³. Ultrastructural examination of negatively stained samples from the peak fraction revealed spheroidal, chitosome-like particles 70–120 nm in diameter. Suspension of these particles produced chitin microfibrils when incubated with uridine diphosphate N-acetylglucosamine, the substrate for chitin synthetase.Non Standard Abbreviations Used GlcNAc N-acetylglucosamine - UDP-GlcNAc uridine diphosphate N-acetylglucosamine - PYG agar 1.25 g of peptone, 1.25 g of yeast extract, 3 g of glucose, and 20 of agar per 1000 ml of water, the pH being adjusted to 6.8 with KOH after autoclaving - EGTA ethyleneglycol-bis (-aminoethylether)-N,N-tetraacetic acid     

The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1.

Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI). This chemically simple step is genetically complex because three genes are required in both mammals and yeast. Mammalian PIG-A and PIG-C are homologous to yeast GPI3 and GPI2, respectively; however, mammalian PIG-H is not homologous to yeast GPI1. Here, we report cloning of a human homolog of GPI1 (hGPI1) and demonstrate that four mammalian gene products form a protein complex in the endoplasmic reticulum membrane. PIG-L, which is involved in the second step in GPI synthesis, GlcNAc-PI de-N-acetylation, did not associate with the isolated complex. The protein complex had GPI-GlcNAc transferase (GPI-GnT) activity in vitro, but did not mediate the second reaction. Bovine PI was utilized approximately 100-fold more efficiently than soybean PI as a substrate, and lyso PI was a very inefficient substrate. These results suggest that GPI-GnT recognizes the fatty acyl chains of PI. The unusually complex organization of GPI-GnT may be relevant to selective usage of PI and/or regulation.     

Formation of dolichol-linked sugar intermediates during the postnatal development of skeletal muscle

The postnatal development of skeletal muscle is characterized by changes in membrane function associated with N-linked glycoproteins. In the present study, early reactions involved in the synthesis of the dolichol-linked core oligosaccharide were examined in neonatal and adult rabbit skeletal muscle sarcoplasmic reticulum membranes. The initial rate of N-acetylglucosamine incorporation in the presence of exogenous dolichol phosphate was similar between neonate and adult (3.5-4.1 pmol of GlcNAc/min/mg). The Km values for UDP-GlcNAc and exogenous dolichol phosphate were similar. Tunicamycin (0.04-0.08 micrograms/ml) inhibited N-acetylglucosamine incorporation by 50%. UDP-GlcNAc pyrophosphatase activity was greater in neonatal membranes than adult (840 versus 350 pmol of GlcNAc-1-P/min/mg), explaining, in part, the greater enhancement of neonatal GlcNAc incorporation by pyrophosphatase inhibitors. Nucleotide-sugar pyrophosphatase inhibitors (alpha, beta-methylene ATP and dimercaptopropanol) increased the capacity of neonatal activity 4-fold and adult enzyme 2-fold. Analysis of dolichol-linked products by mild acid hydrolysis however, revealed that neonate had higher capacity for N,N'-diacetylchitobiosyl(pyro)phosphoryldolichol synthesis than adult. Mannosyltransferase and glucosyltransferase were elevated 6- and 5-fold in neonate compared to adult membranes. Neonate exhibited 4-fold greater GDP-Man pyrophosphatase activity than adult (500 versus 125 pmol of Man-1-P/min/mg). The Km for GDP-Man increased in the presence of exogenous dolichol phosphate. Increasing concentrations of exogenous dolichol phosphate did not equalize neonate and adult mannosyltransferase activity, indicating that the decline in activity during development was not due to a decrease in a pool of dolichol phosphate accessible to mannosyltransferase. Glucosyltransferase for the synthesis of glucosylphosphoryldolichol was also elevated 5-fold in neonatal compared to adult sarcoplasmic reticulum (7 versus 1.4 pmol of Glc/min/mg). In a previous study, it was reported that glycoprotein sialyltransferase activity decreased by a factor of 6.5 during the postnatal maturation and that total membrane hexose content of sarcoplasmic reticulum decreased by a factor of 8. Together, these results suggest that the postnatal development of skeletal muscle is characterized by coordinated changes in the expression of enzymes involved in both the "early" and "late" reactions of N-linked oligosaccharide biosynthesis.     

Enhanced O-GlcNAc protein modification is associated with insulin resistance in GLUT1-overexpressing muscles

O-linked glycosylation on Ser/Thr with single N-acetylglucosamine (O-GlcNAcylation) is a reversible modification of many cytosolic/nuclear proteins, regulated in part by UDP-GlcNAc levels. Transgenic (T) mice that overexpress GLUT1 in muscle show increased basal muscle glucose transport that is resistant to insulin stimulation. Muscle UDP-GlcNAc levels are increased. To assess whether GLUT4 is a substrate for O-GlcNAcylation, we translated GLUT4 mRNA (mutated at the N-glycosylation site) in rabbit reticulocyte lysates supplemented with [(35)S]methionine. O-GlcNAcylated proteins were galactosylated and separated by lectin affinity chromatography; >20% of the translated GLUT4 appeared to be O-GlcNAcylated. To assess whether GLUT4 or GLUT4-associated proteins were O-GlcNAcylated in muscles, muscle membranes were prepared from T and control (C) mice labeled with UDP-[(3)H]galactose and immunoprecipitated with anti-GLUT4 IgG (or nonimmune serum), and N-glycosyl side chains were removed enzymatically. Upon SDS-PAGE, several bands showed consistently two- to threefold increased labeling in T vs. C. Separating galactosylated products by lectin chromatography similarly revealed approximately threefold more O-GlcNAc-modified proteins in T vs. C muscle membranes. RL-2 immunoblots confirmed these results. In conclusion, chronically increased glucose flux, which raises UDP-GlcNAc in muscle, results in enhanced O-GlcNAcylation of membrane proteins in vivo. These may include GLUT4 and/or GLUT4-associated proteins and may contribute to insulin resistance in this model.     

Biosynthesis of CMP-N,N'-diacetyllegionaminic acid from UDP-N,N'-diacetylbacillosamine in Legionella pneumophila

Legionaminic acid is a nine-carbon alpha-keto acid that is similar in structure to other members of the sialic acid family that includes neuraminic acid and pseudaminic acid. It is found as a component of the lipopolysaccharide in several bacterial species and is perhaps best known for its presence in the O-antigen of the causative agent of Legionnaires' disease, Legionella pneumophila. In this work, the enzymes responsible for the biosynthesis and activation of N, N'-diacetyllegionaminic acid are identified for the first time. A cluster of three L. pneumophila genes bearing homology to known sialic acid biosynthetic genes ( neuA,B,C) were cloned and overexpressed in Escherichia coli. The NeuC homologue was found to be a hydrolyzing UDP- N, N'-diacetylbacillosamine 2-epimerase that converts UDP- N, N'-diacetylbacillosamine into 2,4-diacetamido-2,4,6-trideoxymannose and UDP. Stereochemical and isotopic labeling studies showed that the enzyme utilizes a mechanism involving an initial anti elimination of UDP to form a glycal intermediate and a subsequent syn addition of water to generate product. This is similar to the hydrolyzing UDP- N-acetylglucosamine 2-epimerase (NeuC) of sialic acid biosynthesis, but the L. pneumophila enzyme would not accept UDP-GlcNAc as an alternate substrate. The NeuB homologue was found to be a N, N'-diacetyllegionaminic acid synthase that condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate (PEP), although the in vitro activity of the recombinant enzyme (isolated as a MalE fusion protein) was very low. The synthase activity was dependent on the presence of a divalent metal ion, and the reaction proceeded via a C-O bond cleavage process, similar to the reactions catalyzed by the sialic acid and pseudaminic acid synthases. Finally, the NeuA homologue was shown to possess the CMP- N, N'-diacetyllegionaminic acid synthetase activity that generates the activated form of legionaminic acid used in lipopolysaccharide biosynthesis. Together, the three enzymes constitute a pathway that converts a UDP-linked bacillosamine derivative into a CMP-linked legionaminic acid derivative.