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The first step of lipid A biosynthesis in Escherichia coli (E. coli) is catalyzed by LpxA (EcLpxA), an acyltransferase selective for UDP-N-acetylglucosamine (UDP-GlcNAc) and R-3-hydroxymyristoyl-acyl carrier protein (3-OH-C14-ACP), and is an essential step in majority of Gram-negative bacteria. Since the majority of lipid A species isolated from F. novicida contains 3-OH-C16 or 3-OH-C18 at its C3 and C3′ positions, FnLpxA was thought to be selective for longer acyl chain (3-OH-C16 and 3-OH-C18) over short acyl chain (3-OH-C14, 3-OH-C12, and 3-OH-C10). Here we demonstrate that Francisella novicida (F. novicida) lpxA functionally complements an E. coli lpxA knockout mutant and efficiently transfers 3-OH-C14 as well as 3-OH-C16 in E. coli. Our results implicate that the acyl chain length of lipid A is determined by several factors including acyl chain selectivity of LpxA and downstream enzymes, as well as the composition of the acyl-ACP pool in vivo. We also report the crystal structure of F. novicida LpxA (FnLpxA) at 2.06 A. The N-terminal parallel beta-helix (LβH) and C-terminal alpha-helical domain are similar to other reported structures of LpxAs. However, our structure indicates that the supposed ruler residues for hydrocarbon length, 171L in one monomer and 168H in the adjacent monomer in a functional trimer of FnLpxA, are located just 3.8 A apart that renders not enough space for binding of 3-OH-C12 or longer acyl chains. This implicates that FnLpxA may have an alternative hydrophobic pocket, or the acyl chain may bend while binding to FnLpxA. In addition, the FnLpxA structure suggests a potential inhibitor binding site for development of antibiotics.

Hyperglycemia-induced activation of hexosamine biosynthesis pathway (HBP) has been implicated in the development of insulin resistance in skeletal muscles. In the present study, the content of uridine-5'-diphospho-N-acetylglucosamine, the end product of the HBP, was elevated in skeletal muscle of obese diabetic KKA(y) mice, compared with control mice. To elucidate the effect of elevated HBP in the skeletal muscle, we treated C2C12 myoblasts with glucosamine, an intermediate metabolite of the HBP. Glucosamine induced lipid accumulation and significantly increased the mRNA expression levels of peroxisome proliferator-activated receptor gamma, adiponectin, and aP2 in C2C12 myoblasts. Similar mRNA changes were observed in skeletal muscles of Sprague-Dawley rats treated with glucosamine infusion. Our results provide a possible explanation of hyperglycemia-induced insulin resistance in skeletal muscle.

UDP-N-acetylglucosaminyl transferase (OGT) is a key enzyme of a novel signal transduction pathway that regulates protein function through O-linked glycosylation. In the current study, we found that sodium vanadate potently inhibits OGT activity in brain cytosol (IC50 = 55 microM) and nucleosol (IC50 = 150 microM), but fails to alter activity of a related enzyme (UDP-galactosyltransferase). Vanadate also inhibits OGT activity in cytosol (IC50 of 2.3 microM) and nucleosol (IC50 of 130) derived from a stable HeLa cell line that overexpresses OGT. When HeLa cytosol was immunopurified to separate OGT from other cellular proteins, vanadate still inhibited OGT activity (IC50 = 2 microM). We conclude that OGT derived from cytosol exhibits greater vanadate sensitivity than nucleosol OGT and that a large difference exists (25-fold) in vanadate sensitivity when comparing OGT activity in different cell types (IC50 of 55 microM for brain cytosol vs. 2.3 microM for HeLa cytosol). Understanding the mechanism(s) by which a tyrosine phosphatase inhibitor differentially reduces OGT activity should lead to new insights into OGT function and regulation.

Uridine-5′-diphospho-N-acetylglucosamine, when oxidized with periodate to the corresponding aldehyde (o-UDP-GlcNAc), was a potent inhibitor of the methylcoenzyme M methylreductase reaction which catalyzes the reductive demethylation of methylcoenzyme M to methane. The oxidation product, o-UDP-GlcNAc, appears to bind to the UDP-GlcNAc site of the enzyme and inhibits the reduction of methylcoenzyme M both by MRF or its active hydrolytic fragment HS-HTP. The kinetic patterns indicate that o-UDP-GlcNAc inhibition is noncompetitive with HS-HTP or MRF, and the Hill coefficient indicated that there was cooperativity between the UDP and HS-HTP binding sites. The methylreductase enzyme was protected from o-UDP-GlcNAc inhibition by prior exposure to low concentrations of MRF, HS-HTP, at the same concentration as MRF, was not effective in protecting the enzyme from inhibition by o-UDP-GlcNAc.

Streptozotocin has been widely used to create animal models of diabetes. Structurally, streptozotocin resembles N-acetylglucosamine, with a nitrosourea group corresponding to the acetate present in N-acetylglucosamine. Streptozotocin has recently been shown to inhibit O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase, which removes O-linked N-acetylglucosamine from proteins. Compared to other cells, beta-cells express much more of the enzyme O-GlcNAc transferase, which catalyzes addition of O-linked N-acetylglucosamine to proteins. This suggests why beta-cells might be particularly sensitive to streptozotocin. In this report, we demonstrate that both streptozotocin and glucose stimulate O-glycosylation of a 135 kD beta-cell protein. Only the effect of glucose, however, was blocked by inhibition of fructose-6-phosphate amidotransferase, suggesting that glucose acts through the glucosamine pathway to provide UDP-N-acetylglucosamine for p135 O-glycosylation. The fact that both glucose and streptozotocin stimulate p135 O-glycosylation provides a possible mechanism by which hyperglycemia may cause streptozotocin-like effects in beta-cells and thus contribute to the development of type 2 diabetes.

When beef liver microsomes are labeled with UDP-[3H]N-acetyl glucosamine, three different lipid-bound saccharides are labeled: dolichol pyrophosphoryl-GlcNAc, dolichol pyrophosphoryl-(GlcNAc)2 and a previously uncharacterized compound (component III). Incubation with UDP leads to the disappearance of dolichol pyrophosphoryl-(GlcNac)2 from microsomes prelabeled with UDP-3H-N-acetyl glucosamine. This result provides further support for the suggestion of Leloir et al. (6) that dolichol pyrophosphoryl-(GlcNAc)2 is synthesized from dolichol pyrophosphoryl-GlcNAc and UDP-N-acetyl glucosamine. Component III cannot be discharged with UMP or UDP. It elutes from DEAE-cellulose exactly as the other components. The saccharide portion of component III has a molecular weight of 800–900 and contains glucose in addition to N-acetyl glucosamine.

The effect of choline deficiency on the kinetic properties of the microsomal enzyme UDP-glucuronyltransferase (EC2.4.1.17) was investigated in rats. Animals fed choline-deficient diets, as compared with animals fed a choline-replete diet or standard laboratory chow, showed almost a three-fold increase in enzyme activity when the enzyme was assayed at physiological concentrations of UDP-glucuronic acid (0.25 mM). The increase in activity appeared to be due to an enhanced affinity of the enzyme for UDP-glucuronic acid rather than to an increase in the amount of enzyme. These data indicate that the kinetic properties of tightly bound membrane enzymes are altered by a dietary change that is known to cause liver disease in the rat.

Summary: Recent finding of a-N-ocetylglucosamine( I)phospho(6)mannose diesters in lysosomal enzymes suggested that formation of monnose 6-phosphate residues involves transfer of N-acetylglucosamine l- hos hote to mannose. Using dephosphorylated R-hexosaminidase as acceptor and F t3-3 “I P UDP-N-acetylglucosamine OS donor for the phosphate group, phosphorylation of R-hexosaminidose by microsomes from rot liver, humon placenta and human skin fibroblosts wos achieved. The reaction was not affected by tunicomycin. Acid hydrolysis released monnose 6-[32P] phosphate from the phosphorylated l3-hexosaminidase. Our results suggest that lysosomal enzymes are phosphorylated by tronsfer of N-acetylglucosamine l-phosphate from UDP-N-ocetylglucosamine. The transferose activity wos deficient in fibroblasts from patients affected with l-cell disease. This deficiency is proposed to be the primary enzyme defect in I-ccl I disease. peerReviewed

The free nucleotide content of red blood cells from heifers was determined by column chromatography on the day of birth and at 1, 3, 6, and 12 months. Uric acid riboside, which was relatively low on the day of birth, was the major ultraviolet-absorbing compound in the nucleotide pool at 3 months and at the later sampling times. The concentration of UDPglucose, UDP-N-acetylglucosamine, ATP, GTP, and UTP decreased between the day of birth and 1 month and between 1 month and 3 months. The concentrations of these nucleotides were the same at 6 and 12 months as they were at 3 months. The concentrations of NAD, AMP, UMP, and IMP also decreased with time; the concentration of NADP was practically constant.

Addition of the antibiotic bacitracin to a membrane preparation of Saccharomyces cerevisiae enriched in plasma membrane and incubated in vitro with UDP- [ 3 H] GlcNAc, leads to an inhibition of the formation of polyprenyl diphosphate di-N-acetylchitobiose, with a concomitant accumulation of label in polyprenyl diphosphate N-acetylglucosamine. Bacitracin also prevents to a large extent the incorporation of N-acetylglucosamine into a protein fraction.

Summary In the presence of UDP-N-acetyl glucosamine and CDP-glycerol a membrane preparation from Staphylococcus aureus synthesizes a lipophylic compound, which is an intermediate in the synthesis of the linkage region between teichoic acid (polyribitol phosphate) and peptidoglycan. The synthesis of this intermediate is inhibited by Tunicamycin.

Summary Calf-liver rough microsomes incorporated N-acetyl[3H] glucosamine from UDP-[3H]GlcNAc or [14C]mannose from GDP-[14C]Man into products soluble in chloroform-methanol (2:1). The [3H] GlcNAclabeled material had the hydrolytic and chromatographic properties of a polyisoprenyl N-acetylglucosaminyl pyrophosphate whereas the 14C-mannolipid exhibited the characteristics of a polyisoprenyl mannosyl phosphate. Tunicamycin inhibited the production of the [3H]G1cNAc-lipid but did not affect the synthesis of the 14C-mannolipid. This inhibition accounts for the effect of the antibiotic upon glycoprotein formation in vivo since polyprenyl sugar phosphates are intermediates in the biosynthesis of certain glycoproteins.

The biosynthesis of uridine diphosphate N-acetyl-D-mannosaminuronic acid from uridine diphosphate N-acetyl-D-glucosamine occurs in two steps. The enzyme responsible for the first step, the epimerization of uridine diphosphate N-acetyl-D-glucosamine to uridine diphosphate N-acetyl-D-mannosamine, is separated by means of hydroxylapatite chromatography from the enzyme for the second step, the NAD-linked dehydrogenation of uridine diphosphate N-acetyl-D-mannosamine. At equilibrium of the epimerase reaction, the ratio of the glucosamine residue to the mannosamine residue is about 9:1.

Summary The rate of synthesis of N-acetylglucosaminylpyrophosphoryldolichol in dense membrane fractions of rat lung microsomes supplemented with dolichol phosphate was enhanced by the addition of phospholipids. Phosphatidylglycerol alone was more effective than a phospholipid mixture. Only phosphatidylinositol of other phospholipids tested significantly enhanced the reaction rate. The effect was more apparent in dense membrane fractions (rough endoplasmic reticulum) which have a low total phospholipid and phosphatidylglycerol content.

A tunicamycin-like antibiotic 24010 at a concentration of 1 μg/ml selectively inhibited the in vivo synthesis of glycerol teichoic acid of cell walls in Bacillus cereus AHU 1030. Incubation of membranes of this strain with N -acetylglucosaminyl pyrophosphorylundecaprenol and UDP- N -acetylmannosamine led to formation of a glycolipid having a saccharide moiety identical with the cell wall teichoic acid linkage unit, N -acetylmannosaminylβ(1→4)- N -acetylglucosamine. The membranes also catalyzed transfer of glycerol phosphate units from CDP-glycerol to this disaccharide-linked lipid. Thus the biosynthesis of the cell wall glycerol teichoic acid in this strain seems to involve the disaccharide-linked lipid as an intermediate.

Amphomycin, a selective inhibitor of peptidoglycan synthesis of bacteria, inhibited the lipid intermediates accumulation and the peptidoglycan synthesis from UDP-N-acetylmuramyl-L-Ala-D-Glu-[3H]-meso-Dpm-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) and UDP-N-acetylglucosamine (UDP-GlcNAc) with a particulate fraction from Bacillus megaterium KM, and also inhibited the formation of MurNAc (-pentapeptide)-P-P-lipid in the absence of UDP-GlcNAc. But it did not inhibit the formation of peptidoglycan from MurNAc(-pentapeptide)-P-P-lipid and UDP-GlcNAc with the same system of the organism. Thus, it is concluded that the site of action of amphomycin is phospho-MurNAc-pentapeptide translocase in peptidoglycan synthesis.

Bacillus subtilis membranes can transfer either N-acetylmuramyl-pentapeptide phosphate or N-acetylglucosaminyl phosphate from UMP directly onto undecaprenyl phosphate. Tunicamycin blocks only the latter transfer and inhibits peptidoglycan synthesis by toluenized cells of Bacillus megaterium utilizing added nucleotide sugar precursors or cell wall synthesis by intact cells of B. subtilis . Tunicamycin prevents formation of the cell wall disaccharide lipid intermediate by blocking transfer of N-acetylglucosamine onto undecaprenyl muramyl pentapeptidyl pyrophosphate.

An affinity chromatography procedure was developed for isolating antibodies that recognized phosphodiester-linked α- N -acetylglucosamine-1-phosphate (α-GlcNAc-1-P) residues. The affinity resin consisted of uridine-5′-diphospho-α-N-acetylglucosamine (UDPGlcNAc) conjugated to Sepharose. Antiserum prepared against Proteinase 1 from the cellular slime mold, Dictyostelium discoideum was used as a source of the anti-α-GlcNAc-1-P antibodies. Immunoblot assays showed that the affinity-isolated antibodies recognized phosphoglycosylated subunits of Proteinase 1, and that UDPG1cNAc blocked this interaction.