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The hexosamine biosynthetic pathway (HBP) is a glucose metabolism pathway that results in the synthesis of a nucleotide sugar UDP-GlcNAc, which is subsequently used for the post-translational modification (O-GlcNAcylation) of intracellular proteins that regulate nutrient sensing and stress response. The HBP is carried out by a series of enzymes, many of which have been extensively implicated in cancer pathophysiology. Increasing evidence suggests that elevated activation of the HBP may act as a cancer biomarker. Inhibition of HBP enzymes could suppress tumor cell growth, modulate the immune response, reduce resistance, and sensitize tumor cells to conventional cancer therapy. Therefore, targeting the HBP may serve as a novel strategy for treating cancer patients. Here, we review the current findings on the significance of HBP enzymes in various cancers and discuss future approaches for exploiting HBP inhibition for cancer treatment.

The mechanistic target of rapamycin (mTOR) controls metabolic pathways in response to nutrients. Recently, we have shown that mTOR complex 2 (mTORC2) modulates the hexosamine biosynthetic pathway (HBP) by promoting the expression of the key enzyme of the HBP, glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1). Here, we found that GFAT1 Ser-243 phosphorylation is also modulated in an mTORC2-dependent manner. In response to glutamine limitation, active mTORC2 prolongs the duration of Ser-243 phosphorylation, albeit at lower amplitude. Blocking glycolysis using 2-deoxyglucose robustly enhances Ser-243 phosphorylation, correlating with heightened mTORC2 activation, increased AMPK activity, and O-GlcNAcylation. However, when 2-deoxyglucose is combined with glutamine deprivation, GFAT1 Ser-243 phosphorylation and mTORC2 activation remain elevated, whereas AMPK activation and O-GlcNAcylation diminish. Phosphorylation at Ser-243 promotes GFAT1 expression and production of GFAT1-generated metabolites including ample production of the HBP end-product, UDP-GlcNAc, despite nutrient starvation. Hence, we propose that the mTORC2-mediated increase in GFAT1 Ser-243 phosphorylation promotes flux through the HBP to maintain production of UDP-GlcNAc when nutrients are limiting. Our findings provide insights on how the HBP is reprogrammed via mTORC2 in nutrient-addicted cancer cells.

Psychrobacter cryohalolentis strain K5T is a Gram-negative organism first isolated in 2006. It has a complex O-antigen that contains, in addition to l-rhamnose and d-galactose, two diacetamido- and a triacetamido-sugar. The biochemical pathways for the production of these unusual sugars are presently unknown. Utilizing the published genome sequence of the organism, we hypothesized that the genes 0612, 0638, and 0637 encode for a 4,6-dehydratase, an aminotransferase, and an N-acetyltransferase, respectively, which would be required for the biosynthesis of one of the diacetamido-sugars, 2,4-diacetamido-2,4,6-trideoxy-d-glucose, starting from UDP-N-acetylglucosamine. Here we present functional and structural data on the proteins encoded by the 0638 and 0637 genes. The kinetic properties of these enzymes were investigated by a discontinuous HPLC assay. An X-ray crystallographic structure of 0638, determined in its external aldimine form to 1.3 Å resolution, demonstrated the manner in which the UDP ligand is positioned into the active site. It is strikingly different from that previously observed for PglE from Campylobacter jejuni, which functions on the same substrate. Four X-ray crystallographic structures were also determined for 0637 in various complexed states at resolutions between 1.3 and 1.55 Å. Remarkably, a tetrahedral intermediate mimicking the presumed transition state was trapped in one of the complexes. The data presented herein confirm the hypothesized functions of these enzymes and provide new insight into an unusual sugar biosynthetic pathway in Gram-negative bacteria. We also describe an efficient method for acetyl-CoA synthesis that allowed us to overcome its prohibitive cost for this analysis.

Embryonic and adult stem cells possess the capability of self-renewal and lineage-specific differentiation. The intricate balance between self-renewal and differentiation is governed by developmental signals and cell-type-specific gene regulatory mechanisms. A perturbed intra/extracellular environment during lineage specification could affect stem cell fate decisions resulting in pathology. Growing evidence demonstrates that metabolic pathways govern epigenetic regulation of gene expression during stem cell fate commitment through the utilization of metabolic intermediates or end products of metabolic pathways as substrates for enzymatic histone/DNA modifications. UDP-GlcNAc is one such metabolite that acts as a substrate for enzymatic mono-glycosylation of various nuclear, cytosolic, and mitochondrial proteins on serine/threonine amino acid residues, a process termed protein O-GlcNAcylation. The levels of GlcNAc inside the cells depend on the nutrient availability, especially glucose. Thus, this metabolic sensor could modulate gene expression through O-GlcNAc modification of histones or other proteins in response to metabolic fluctuations. Herein, we review evidence demonstrating how stem cells couple metabolic inputs to gene regulatory pathways through O-GlcNAc-mediated epigenetic/transcriptional regulatory mechanisms to govern self-renewal and lineage-specific differentiation programs. This review will serve as a primer for researchers seeking to better understand how O-GlcNAc influences stemness and may catalyze the discovery of new stem-cell-based therapeutic approaches.

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.

Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata. In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAPs, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2, whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAPs responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval-larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that LdUAP1 and LdUAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata.

Neisseria meningitidis serogroup A non-hydrolyzing uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (NmSacA) catalyzes the interconversion between UDP-GlcNAc and uridine 5'-diphosphate-N-acetylmannosamine (UDP-ManNAc). It is a key enzyme involved in the biosynthesis of the capsular polysaccharide [-6ManNAcα1-phosphate-]n of N. meningitidis serogroup A, one of the six serogroups (A, B, C, W-135, X, and Y) that account for most cases of N. meningitidis-caused bacterial septicemia and meningitis. N. meningitidis serogroup A is responsible for large epidemics in the developing world, especially in Africa. Here we report that UDP-ManNAc could be used as a substrate for C-terminal His6-tagged recombinant NmSacA (NmSacA-His6) in the absence of UDP-GlcNAc. NmSacA-His6 was activated by UDP-GlcNAc and inhibited by 2-acetamidoglucal and UDP. Substrate specificity study showed that NmSacA-His6 could tolerate several chemoenzymatically synthesized UDP-ManNAc derivatives as substrates although its activity was much lower than non-modified UDP-ManNAc. Homology modeling and molecular docking revealed likely structural determinants of NmSacA substrate specificity. This is the first detailed study of N. meningitidis serogroup A UDP-GlcNAc 2-epimerase.

Aminosugars produced in the hexosamine pathway (HP) are utilized in protein glycosylation reactions involved in protein maturation and cellular signaling. Recent evidence revealed a role of the HP in protein quality control and ageing. Elevation of the HP product UDP-N-acetylglucosamine in the nematode Caenorhabditis elegans results in resistance towards toxic aggregation-prone proteins, and extended lifespan. Glutamine-fructose 6 phosphate aminotransferase (GFAT-1), the HP's key enzyme, is a target of the unfolded protein response (UPR). Thus, cardiac stress in mice results in GFAT-1 activation that triggers a cytoprotective response. Feeding of glucosamine to aged mice increases their life expectancy. Here we discuss HP activation and cellular protein quality control mechanisms that result in stress resistance and suppression of age-related proteotoxicity.

The emergence of antibiotic-resistant strains of pathogenic bacteria is an increasing threat to global health that underscores an urgent need for an expanded antibacterial armamentarium. Gram-negative bacteria, such as Escherichia coli, have become increasingly important clinical pathogens with limited treatment options. This is due in part to their lipopolysaccharide (LPS) outer membrane components, which dually serve as endotoxins while also protecting Gram-negative bacteria from antibiotic entry. The LpxC enzyme catalyzes the committed step of LPS biosynthesis, making LpxC a promising target for new antibacterials. Here, we present the first structure of an LpxC enzyme in complex with the deacetylation reaction product, UDP-(3-O-(R-3-hydroxymyristoyl))-glucosamine. These studies provide valuable insight into recognition of substrates and products by LpxC and a platform for structure-guided drug discovery of broad spectrum Gram-negative antibiotics.

LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.

Human milk contains high amounts of complex oligosaccharides, which can be utilized especially by Bifidobacterium species in the infant gut as a carbon and energy source. N-acetyl-d-glucosamine is a building block of these oligosaccharides, and molecular details on the release and utilization of this monosaccharide are not fully understood. In this work we have studied some of the enzymatic properties of three N-acetyl-β-D-hexosaminidases encoded by the genome of the intestinal isolate Bifidobacterium longum subsp. infantis ATCC 15697 and the gene expression of the corresponding genes during bacterial growth on human milk oligosaccharides. These enzymes belong to the glycosyl hydrolase family 20, with several homologs in bifidobacteria. Their optimum pH was 5.0 and optimum temperature was 37 °C. The three enzymes were active on the GlcNAcβ1-3 linkage found in lacto-N-tetraose, the most abundant human milk oligosaccharide. Blon_0459 and Blon_0732, but not Blon_2355, cleaved branched GlcNAcβ1-6 linkages found in lacto-N-hexaose, another oligosaccharide abundant in breast milk. Bifidobacterium infantis N-acetyl-β-D-hexosaminidases were induced during early growth in vitro on human milk oligosaccharides, and also during growth on lacto-N-tetraose or lacto-N-neotetraose. The up-regulation of enzymes that convert this monosaccharide into UDP-N-acetylglucosamine by human milk oligosaccharides suggested that this activated sugar is used in peptidoglycan biosynthesis. These results emphasize the complexity of human milk oligosaccharide consumption by this infant intestinal isolate, and provide new clues into this process.

The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (∼65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.

The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked β-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1–14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017–1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G1 cells, declines throughout the S phase, increases again during late S/early G2, and persists through late G2 and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression. (SFI) Principal Investigator award (07/IN1/B958) and a Health Research Board (HRB) Project Grant (RP/2005/265) (to N. F. L.). Deposited by bulk import

Nucleoside diphosphate sugars serve in essential roles in metabolic processes. They have, therefore, been used in mechanistic studies on glycosylation reactions, and their analogues have been synthesised as enzyme and receptor inhibitors. Despite extensive biochemical research, little is known about their chemical reactions. In the present work the chemical cleavage of two different types of nucleoside diphosphate sugars has been studied. UDP-Glc is phosphorylated at the anomeric carbon, whereas in ADP-Rib C-1 is unsubstituted, allowing hence the equilibrium between cyclic hemiacetal and acyclic carbonyl forms. Due to the structural difference, these substrates react via different pathways under slightly alkaline conditions: while UDP-Glc reacts exclusively by a nucleophilic attack of a glucose hydroxyl group on the diphosphate moiety, ADP-Rib undergoes a complex reaction sequence that involves isomerisation processes of the acyclic ribose sugar and results in a release of ADP.

The molecular weight of hyaluronan is important for its rheological and biological function. The molecular mechanisms underlying chain termination and hence molecular weight control remain poorly understood, not only for hyaluronan synthases but also for other beta-polysaccharide synthases, e.g. cellulose, chitin, and 1,3-betaglucan synthases. In this work, we manipulated metabolite concentrations in the hyaluronan pathway by overexpressing the five genes of the hyaluronan synthesis operon in Streptococcus equi subsp. zooepidemicus. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased molecular weight, whereas overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased molecular weight. The highest molecular mass observed was at 3.4 +/- 0.1 MDa twice that observed in the wild-type strain, 1.8 +/- 0.1 MDa. The data indicate that (a) high molecular weight is achieved when an appropriate balance of UDP-N-acetylglucosamine and UDP-glucuronic acid is achieved, (b) UDP-N-acetylglucosamine exerts the dominant effect on molecular weight, and (c) the wild-type strain has suboptimal levels of UDP-N-acetylglucosamine. Consistent herewith molecular weight correlated strongly (rho = 0.84, p = 3 x 10(-5)) with the concentration of UDP-N-acetylglucosamine. Data presented in this paper represent the first model for hyaluronan molecular weight control based on the concentration of activated sugar precursors. These results can be used to engineer strains producing high molecular weight hyaluronan and may provide insight into similar polymerization mechanisms in other polysaccharides.

Identifying direct targets of kinases and determining how their activities are regulated are central to understanding how they generate biological responses. Genetic and biochemical studies have shown that Mycobacterium tuberculosis serine/threonine protein kinases PknA and PknB play a role in modulating cell shape and possibly cell division. In this report, we show that the enzyme N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) of M. tuberculosis is a novel substrate of PknB and is phosphorylated on threonine residues. GlmU carries out two important biochemical activities: a C-terminal domain catalyzes the transfer of acetyl group from acetyl coenzyme A to glucosamine-1-phosphate to produce N-acetylglucosamine-1-phosphate, which is converted into UDP-N-acetylglucosamine by the transfer of uridine 5'-monophosphate (from uridine 5'-triphosphate), a reaction catalyzed by the N-terminal domain. We determined the crystal structures of GlmU in apo form and UDP-N-acetylglucosamine-bound form, and analyzed them to identify threonine residues that may be accessible to PknB. The structure shows a two-domain architecture, with an N-terminal domain having an alpha/beta-like fold and with a C-terminal domain that forms a left-handed parallel beta-helix structure. Kinase assays with PknB using the N- and C-terminal domains of GlmU as substrates illustrated that PknB phosphorylates GlmU in the C-terminal domain. Furthermore, mutational studies reveal one of the five threonines present in region 414-439 to be phosphorylated by PknB. Structural and biochemical analyses have shown the significance of a variable C-terminal tail in regulating acetyltransferase activity. Notably, we demonstrate that although PknB-mediated phosphorylation of GlmU does not affect its uridyltransferase activity, it significantly modulates the acetyltransferase activity. These findings imply a role for PknB in regulating peptidoglycan synthesis by modulating the acetyltransferase activity of GlmU.

UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5'' stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-hexos-4-ulose. PseB catalyzes the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylori. PseB is proposed to use its tightly bound NADP+ to oxidize UDP-GlcNAc at C-4'', enabling dehydration. The alpha,beta unsaturated ketone intermediate is then reduced by delivery of the hydride to C-6'' and a proton to C-5''. Consistent with this, PseB from C. jejuni has been found to incorporate deuterium into the C-5'' position of product during catalysis in D2O. Likewise, PseB catalyzes solvent isotope exchange into the H-5'' position of product, and eliminates HF from the alternate substrate, UDP-6-deoxy-6-fluoro-GlcNAc. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.

Summary The solution structure of Alg13, the glycosyl donor-binding domain of an important bipartite glycosyltransferase in the yeast Saccharomyces cerevisiae , is presented. This glycosyltransferase is unusual in that it is active only in the presence of a binding partner, Alg14. Alg13 is found to adopt a unique topology among glycosyltransferases. Rather than the conventional Rossmann fold found in all GT-B enzymes, the N-terminal half of the protein is a Rossmann-like fold with a mixed parallel and antiparallel β sheet. The Rossmann fold of the C-terminal half of Alg13 is conserved. However, although conventional GT-B enzymes usually possess three helices at the C terminus, only two helices are present in Alg13. Titration of Alg13 with both UDP-GlcNAc, the native glycosyl donor, and a paramagnetic mimic, UDP-TEMPO, shows that the interaction of Alg13 with the sugar donor is primarily through the residues in the C-terminal half of the protein.

Nucleotide-sugar transporters (NSTs) transport activated sugars (e.g. UDP-GlcNAc) from the cytosol to the lumen of the endoplasmic reticulum or Golgi apparatus where they are used to make glycoproteins and glycolipids. UDP-Glc is an important component of the N-glycan-dependent quality control (QC) system for protein folding. Because Entamoeba has this QC system while Giardia does not, we hypothesized that transfected Giardia might be used to identify the UDP-Glc transporter of Entamoeba. Here we show Giardia membranes transport UDP-GlcNAc and have apyrases, which hydrolyze nucleoside-diphosphates to make the antiporter nucleoside-monophosphate. The only NST of Giardia (GlNst), which we could identify, transports UDP-GlcNAc in transfected Saccharomyces and is present in perinuclear and peripheral vesicles and increases in expression during encystation. Entamoeba membranes transport three nucleotide-sugars (UDP-Gal, UDP-GlcNAc, and UDP-Glc), and Entamoeba has three NSTs, one of which has been shown previously to transport UDP-Gal (EhNst1). Here we show recombinant EhNst2 transports UDP-Glc in transfected Giardia, while recombinant EhNst3 transports UDP-GlcNAc in transfected Saccharomyces. In summary, all three NSTs of Entamoeba and the single NST of Giardia have been molecularly characterized, and transfected Giardia provides a new system for testing heterologous UDP-Glc transporters.

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

Autoimmunity is a complex trait disease where the environment influences susceptibility to disease by unclear mechanisms. T cell receptor clustering and signaling at the immune synapse, T cell proliferation, CTLA-4 endocytosis, T(H)1 differentiation, and autoimmunity are negatively regulated by beta1,6GlcNAc-branched N-glycans attached to cell surface glycoproteins. Beta1,6GlcNAc-branched N-glycan expression in T cells is dependent on metabolite supply to UDP-GlcNAc biosynthesis (hexosamine pathway) and in turn to Golgi N-acetylglucosaminyltransferases Mgat1, -2, -4, and -5. In Jurkat T cells, beta1,6GlcNAc-branching in N-glycans is stimulated by metabolites supplying the hexosamine pathway including glucose, GlcNAc, acetoacetate, glutamine, ammonia, or uridine but not by control metabolites mannosamine, galactose, mannose, succinate, or pyruvate. Hexosamine supplementation in vitro and in vivo also increases beta1,6GlcNAc-branched N-glycans in naïve mouse T cells and suppresses T cell receptor signaling, T cell proliferation, CTLA-4 endocytosis, T(H)1 differentiation, experimental autoimmune encephalomyelitis, and autoimmune diabetes in non-obese diabetic mice. Our results indicate that metabolite flux through the hexosamine and N-glycan pathways conditionally regulates autoimmunity by modulating multiple T cell functionalities downstream of beta1,6GlcNAc-branched N-glycans. This suggests metabolic therapy as a potential treatment for autoimmune disease.

Chondroitin polymerase from Escherichia coli strain K4 (K4CP) synthesizes chondroitin (CH) polysaccharides by the alternate addition of N-acetyl- d -galactosamine (GalNAc) and d -glucuronic acid (GlcA) to acceptor CH oligosaccharides in the presence of Mn2+ ions. In this study, we applied matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI–TOF MS) for the further characterization of the products synthesized by K4CP from CH hexasaccharide as an initial acceptor and UDP-GalNAc and UDP-GlcA as donors. The analysis identified individual CH chains of various lengths and enabled the calculation of their average molecular weights. The ion peaks of the CH chains synthesized in the short-time reactions demonstrated not only the alternate addition of GlcA and GalNAc but also the more frequent transfer of GlcA and GalNAc, consistent with our previous kinetic data. In contrast, the MS spectra of the chains synthesized in the long-time reaction showed that CH chains containing GalNAc at the nonreducing ends were more abundant than those containing GlcA. We found that this inconsistency was due to the preferential decomposition of UDP-GlcA by Mn2+ ions. We defined the optimal conditions to yield further elongation of the CH chains that have nearly equal numbers of GlcA and GalNAc residues at the nonreducing ends.

The direct oxidation of UDP-alpha-d-glucose and UDP-N-acetyl-alpha-d-glucosamine to the corresponding uronic acids was explored using either TEMPO or platinum-catalysed oxidation with molecular oxygen. Whilst TEMPO-based procedures gave rise to substantial over-oxidation and/or degradation of UDP-glucose, oxidation of UDP-N-acetyl-glucosamine to UDP-N-acetyl-glucosaminuronic acid was achieved with >90% conversion and ca. 65% isolated yield using a platinum-catalysed procedure.

It has been shown that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have not been identfied. Glutamine: fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP) and activation of this pathway has been shown to be cardioprotective. Glutamine is required for metabolism of glucose via GFAT; therefore, the goal of this study was to determine whether glutamine cardioprotection could be attributed to increased flux through the HBP and elevated levels of O-linked N-acetyl-glucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5mM glucose, and global, no-flow ischemia was induced for 20 minutes followed by 60 minutes of reperfusion. 30 minute pre-treatment with 2.5 mM glutamine significantly improved functional recovery (RPP: 15.6±5.7% Vs. 59.4±6.1%; p

The large cytotoxins of Clostridia species glycosylate and thereby inactivate small GTPases of the Rho family. Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin use UDP-glucose as the donor for glucosylation of Rho/Ras GTPases. In contrast, alpha-toxin from Clostridium novyi N-acetylglucosaminylates Rho GTPases by using UDP-N-acetylglucosamine as a donor substrate. Based on the crystal structure of C. difficile toxin B, we studied the sugar donor specificity of the toxins by site-directed mutagenesis. The changing of Ile-383 and Gln-385 in toxin B to serine and alanine, respectively, largely increased the acceptance of UDP-N-acetylglucosamine as a sugar donor for modification of RhoA. The K(m) value was reduced from 960 to 26 mum for the double mutant. Accordingly, the potential of the double mutant of toxin B to hydrolyze UDP-N-acetylglucosamine was higher than that for UDP-glucose. The changing of Ile-383 and Gln-385 in the lethal toxin of C. sordellii allowed modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduced the acceptance of UDP-glucose as a donor for glycosylation. Vice versa, the changing of the equivalent residues in C. novyi alpha-toxin from Ser-385 and Ala-387 to isoleucine and glutamine, respectively, reversed the donor specificity of the toxin from UDP-N-acetylglucosamine to UDP-glucose. These data demonstrate that two amino acid residues are crucial for the co-substrate specificity of clostridial glycosylating toxins.

The transport of nucleotide sugars from the cytoplasm into the Golgi apparatus is mediated by specialized type III proteins, the nucleotide sugar transporters (NSTs). Transport assays carried out in vitro with Golgi vesicles from mammalian cells showed specific uptake for a total of eight nucleotide sugars. When this study was started, NSTs with transport activities for all but two nucleotide sugars (UDP-Xyl and UDP-Glc) had been cloned. Aiming at identifying these elusive NSTs, bioinformatic methods were used to display putative NST sequences in the human genome. Ten open reading frames were identified, cloned, and heterologously expressed in yeast. Transport capabilities for UDP-Glc and UDP-Xyl were determined with Golgi vesicles isolated from transformed cells. Although a potential UDP-Glc transporter could not be identified due to the high endogenous transport background, the measurement of UDP-Xyl transport was possible on a zero background. Vesicles from yeast cells expressing the human gene SLC35B4 showed specific uptake of UDP-Xyl, and subsequent testing of other nucleotide sugars revealed a second activity for UDP-GlcNAc. Expression of the epitope-tagged SLC35B4 in mammalian cells demonstrated strict Golgi localization. Because decarboxylation of UDP-GlcA is known to produce UDP-Xyl directly in the endoplasmic reticulum and Golgi lumen, our data demonstrate that two ways exist to deliver UDP-Xyl to the Golgi apparatus.

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent enzyme that catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-(R-hydroxymyristoyl)glucosamine and acetate. The structural similarity of the active site of LpxC to metalloproteases led to the proposal that LpxC functions via a metalloprotease-like mechanism. The pH dependence of k(cat)/Km catalyzed by Escherichia coli and Aquifex aeolicus LpxC displayed a bell-shaped curve (EcLpxC yields apparent pKa values of 6.4+/-0.1 and 9.1+/-0.1), demonstrating that at least two ionizations are important for maximal activity. Metal substitution and mutagenesis experiments suggest that the basic limb of the pH profile is because of deprotonation of a zinc-coordinated group such as the zinc-water molecule, whereas the acidic limb of the pH profile is caused by protonation of either Glu78 or His265. Furthermore, the magnitude of the activity decreases and synergy observed for the active site mutants suggest that Glu78 and His265 act as a general acid-base catalyst pair. Crystal structures of LpxC complexed with cacodylate or palmitate demonstrate that both Glu78 and His265 hydrogen-bond with the same oxygen atom of the tetrahedral intermediate and the product carboxylate. These structural features suggest that LpxC catalyzes deacetylation by using Glu78 and His265 as a general acid-base pair and the zinc-bound water as a nucleophile.

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-galactose 4'-epimerase (GALE) interconverts UDP-galactose and UDP-glucose in the final step of the Leloir pathway. Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. The basis of this differential substrate specificity has remained obscure. Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity. We report here a direct test of this hypothesis. In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE. While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate. All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine. The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis. In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates. In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine. Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.

During the establishment of a bacterial infection, the surface molecules of the host organism are of particular importance, since they mediate the first contact with the pathogen. In Caenorhabditis elegans, mutations in the srf-3 locus confer resistance to infection by Microbacterium nematophilum, and they also prevent biofilm formation by Yersinia pseudotuberculosis, a close relative of the bubonic plague agent Yersinia pestis. We cloned srf-3 and found that it encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi apparatus membrane. srf-3 is exclusively expressed in secretory cells, consistent with its proposed function in cuticle/surface modification. We demonstrate that SRF-3 can function as a nucleotide sugar transporter in heterologous in vitro and in vivo systems. UDP-galactose and UDP-N-acetylglucosamine are substrates for SRF-3. We propose that the inability of Yersinia biofilms and M. nematophilum to adhere to the nematode cuticle is due to an altered glycoconjugate surface composition of the srf-3 mutant.

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.

LpxA of Escherichia coli catalyzes the acylation of the glucosamine 3-OH group of UDP-GlcNAc, using R-3-hydroxymyristoyl-acyl carrier protein (ACP) as the donor substrate. We now demonstrate that LpxA in cell extracts of Mesorhizobium loti and Leptospira interrogans, which synthesize lipid A molecules containing 2,3-diamino-2,3-dideoxy-d-glucopyranose (GlcN3N) units in place of glucosamine, do not acylate UDP-GlcNAc. Instead, these LpxA acyltransferases require a UDP-Glc-NAc derivative (designated UDP 2-acetamido-3-amino-2,3-dideoxy-α-d-glucopyranose or UDP-GlcNAc3N), characterized in the preceding paper (Sweet, C. R., Ribeiro, A. A., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25400–25410), in which an amine replaces the glucosamine 3-OH group. L. interrogans LpxA furthermore displays absolute selectivity for 3-hydroxylauroyl-ACP as the donor, whereas M. loti LpxA functions almost equally well with 10-, 12-, and 14-carbon 3-hydroxyacyl-ACPs. The substrate selectivity of L. interrogans LpxA is consistent with the structure of L. interrogans lipid A. The mechanism of L. interrogans LpxA appears to be similar to that of E. coli LpxA, given that the essential His125 residue of E. coli LpxA is conserved and is also required for acyltransferase activity in L. interrogans. Acidithiobacillus ferrooxidans (an organism that makes lipid A molecules containing both GlcN and GlcN3N) has an ortholog of LpxA that is selective for UDP-GlcNAc3N, but the enzyme also catalyzes the acylation of UDP-GlcNAc at a slow rate. E. coli LpxA acylates UDP-GlcNAc and UDP-GlcNAc3N at comparable rates in vitro. However, UDP-GlcNAc3N is not synthesized in vivo, because E. coli lacks gnnA and gnnB. When the latter are supplied together with A. ferrooxidans lpxA, E. coli incorporates a significant amount of GlcN3N into its lipid A.

The critical step for sorting of lysosomal enzymes is the recognition by a Golgi-located phosphotransferase. The topogenic structure common to all lysosomal enzymes essential for this recognition is still not well defined, except that lysine residues seem to play a critical role. Here we have substituted surface-located lysine residues of lysosomal arylsulfatases A and B. In lysosomal arylsulfatase A only substitution of lysine residue 457 caused a reduction of phosphorylation to 33% and increased secretion of the mutant enzyme. In contrast to critical lysines in various other lysosomal enzymes, lysine 457 is not located in an unstructured loop region but in a helix. It is not strictly conserved among six homologous lysosomal sulfatases. Based on three-dimensional structure comparison, lysines 497 and 507 in arylsulfatase B are in a similar position as lysine 457 of arylsulfatase A. Also, the position of oligosaccharide side chains phosphorylated in arylsulfatase A is similar in arylsulfatase B. Despite the high degree of structural homology between these two sulfatases substitution of lysines 497 and 507 in arylsulfatase B has no effect on the sorting and phosphorylation of this sulfatase. Thus, highly homologous lysosomal arylsulfatases A and B did not develop a single conserved phosphotransferase recognition signal, demonstrating the high variability of this signal even in evolutionary closely related enzymes.

N-Acetyl-l-fucosamine is a constituent of surface polysaccharide structures of Pseudomonas aeruginosa and Staphylococcus aureus. The three P. aeruginosa enzymes WbjB, WbjC, and WbjD, as well as the S. aureus homologs Cap5E, Cap5F, and Cap5G, involved in the biosynthesis of N-acetyl-l-fucosamine have been overexpressed and purified to near homogeneity. Capillary electrophoresis (CE), mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy have been used to elucidate the biosynthesis pathway, which proceeds in five reaction steps. WbjB/Cap5E catalyzed 4,6-dehydration of UDP-N-acetyl-d-glucosamine and 3- and 5-epimerization to yield a mixture of three keto-deoxy-sugars. The third intermediate compound was subsequently reduced at C-4 to UDP-2-acetamido-2,6-dideoxy-l-talose by WbjC/Cap5F. Incubation of UDP-2-acetamido-2,6-dideoxy-l-talose (UDP-TalNAc) with WbjD/Cap5G resulted in a new peak separable by CE that demonstrated identical mass and fragmentation patterns by CE-MS/MS to UDP-TalNAc. These results are consistent with WbjD/Cap5G-mediated 2-epimerization of UDP-TalNAc to UDP-FucNAc. A nonpolar gene knockout of wbjB, the first of the genes associated with this pathway, was constructed in P. aeruginosa serotype O11 strain PA103. The corresponding mutant produced rough lipopolysaccharide devoid of B-band O antigen. This lipopolysaccharide deficiency could be complemented with P. aeruginosa wbjB or with the S. aureus homolog cap5E. Insertional inactivation of either the cap5G or cap5F genes abolished capsule polysaccharide production in the S. aureus strain Newman. Providing the appropriate gene in trans, thereby complementing these mutants, fully restored the capsular polysaccharide phenotype.

Protein glycosylation is a common and important process that can alter the stability, half-life, biological activity and receptor recognition of target molecules. We have identified a new putative mouse UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family member, termed GalNAc-T10/ppGaNTase-T10 (gene symbol Galnt10), and determined its expression pattern in mouse CNS using in situ hybridization analysis. Results demonstrated predominant expression of Galnt10 in several distinct hypothalamic, thalamic and amygdaloid nuclei. The most abundant hybridization levels were observed in the paraventricular, ventromedial and arcuate nuclei of the hypothalamus, the anterodorsal and parafascicular nuclei of the thalamus and the central, basomedial and medial nuclei of the amygdala. Expression of Galnt10 was also detected in cerebral cortex, lateral septum, habenula and hippocampus. The localization of this putative glycosyltransferase in distinct regions within the CNS indicates the specificity for complex protein modifications and suggests that region-specific glycosylation represents an essential process in basic biological functions.

6''-Azido-6''-deoxy-UDP-N-acetylglucosamine (UDP-6Az-GlcNAc) is a potential alternate substrate for N-acetylglucosaminyltransferases. This compound could be used to generate various glycoconjugates bearing an azide functionality that could in turn be subjected to further modification using Staudinger ligation or Huisgen cycloaddition. UDP-6Az-GlcNAc is synthesized from α-benzyl-N-acetylglucosaminoside in seven-steps with an overall yield of 6%. It is demonstrated to serve as a substrate donor for the glycosyl transfer reaction catalyzed by the human UDP-GlcNAc:polypeptidyltransferase (OGT) to the acceptor protein nucleoporin 62 (nup62).

Frontal affinity chromatography coupled online to mass spectrometry (FAC/MS) has previously been used to estimate binding constants for individual protein ligands present in mixtures of compounds. In this study FAC/MS is used to determine enzyme substrate kinetic parameters and binding constants for enzyme inhibitors. Recombinant human N-acetylglucosaminyltransferase V was biotinylated and adsorbed onto immobilized streptavidin in a microcolumn (20 microL). The enzyme was shown to be catalytically competent transferring GlcNAc from the donor UDP-GlcNAc to beta-d-GlcpNAc-(1-->2)-alpha-d-Manp-(1-->6)-beta-d-Glcp-OR acceptor giving beta-d-GlcpNAc-(1-->2)-[beta-d-GlcpNAc-(1-->6)]-alpha-d-Manp-(1-->6)-beta-d-Glcp-OR as the reaction product. The kinetic parameters K(m) and V(max) for the immobilized enzyme could be determined by FAC/MS and were comparable to those measured in solution. Analysis of a mixture of eight trisaccharide analogs in a single run yielded K(d) values for each of the eight compounds ranging from 0.3 to 36 microM. These K(d) values were 2 to 10 times lower than the inhibition constants, K(I)'s, determined in solution using a standard radiochemical assay. However, the ranking order of K(d)'s was the same as the ranking of K(I) values. FAC/MS assays can therefore be employed for the rapid estimation of inhibitor K(d) values making it a valuable tool for enzyme inhibitor evaluations.

"French type" sialuria, a presumably dominant disorder that, until now, had been documented in only five patients, manifests with mildly coarse facies, slight motor delay, and urinary excretion of large quantities (1 g/d) of free N-acetylneuraminic acid (NeuAc). The basic defect consists of the very rare occurrence of failed feedback inhibition of a rate-limiting enzyme, in this case uridinediphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase, by a downstream product, in this case cytidine monophosphate (CMP)-NeuAc. We report a new patient with sialuria who has a heterozygous G--A substitution in nucleotide 848 of the epimerase gene, which results in an R266Q change. The proband's other allele, as expected, had no mutation. However, the heterozygous R266Q mutation was detected in the patient's mother, who has similarly increased urinary levels of free NeuAc, thereby confirming, for the first time, the dominant mode of inheritance of this inborn error. The biochemical diagnosis of the proband was verified by the greatly increased level of free NeuAc in his cultured fibroblasts, the NeuAc distribution, mainly (59%) in the cytoplasm, and by the complete failure of 100 microM CMP-NeuAc to inhibit UDP-GlcNAc 2-epimerase activity in the mutant cells. These findings call for expansion of the phenotype to include adults and for more-extensive assaying of free NeuAc in the urine of children with mild developmental delay. The prevalence of sialuria is probably grossly underestimated.

The yeast enzymes involved in UDP-GlcNAc biosynthesis are potential targets for antifungal agents. GNA1, a novel member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, participates in UDP-GlcNAc biosynthesis by catalyzing the formation of GlcNAc6P from AcCoA and GlcN6P. We have solved three crystal structures corresponding to the apo Saccharomyces cerevisiae GNA1, the GNA1-AcCoA, and the GNA1-CoA-GlcNAc6P complexes and have refined them to 2.4, 1.3, and 1.8 A resolution, respectively. These structures not only reveal a stable, β-intertwined, dimeric assembly with the GlcNAc6P binding site located at the dimer interface but also shed light on the catalytic machinery of GNA1 at an atomic level. Hence, they broaden our understanding of structural features required for GNAT activity, provide structural details for related aminoglycosideN-acetyltransferases, and highlight the adaptability of the GNAT superfamily members to acquire various specificities.

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 homogeneous 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 microM and k(cat) of 0.36 s(-1), compared to 2 microM and 1.5 s(-1) for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC(50)s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential bacterial enzyme with both an acetyltransferase and a uridyltransferase activity which have been mapped to the C-terminal and N-terminal domains, respectively. GlmU performs the last two steps in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential precursor in both the peptidoglycan and the lipopolysaccharide metabolic pathways. GlmU is therefore an attractive target for potential antibiotics. Knowledge of its three-dimensional structure would provide a basis for rational drug design. We have determined the crystal structures of Streptococcus pneumoniae GlmU (SpGlmU) in apo form at 2.33 A resolution, and in complex with UDP-N-acetyl glucosamine and the essential co-factor Mg(2+) at 1.96 A resolution. The protein structure consists of an N-terminal domain with an alpha/beta-fold, containing the uridyltransferase active site, and a C-terminal domain with a long left-handed beta-sheet helix (LbetaH) domain. An insertion loop containing the highly conserved sequence motif Asn-Tyr-Asp-Gly protrudes from the left-handed beta-sheet helix domain. In the crystal, S. pneumoniae GlmU forms exact trimers, mainly through contacts between left-handed beta-sheet helix domains. UDP-N-acetylglucosamine and Mg(2+) are bound at the uridyltransferase active site, which is in a closed form. We propose a uridyltransferase mechanism in which the activation energy of the double negatively charged phosphorane transition state is lowered by charge compensation of Mg(2+) and the side-chain of Lys22.

Chitin is an essential cell wall component, synthesis of which is regulated throughout the cell cycle in the yeast Saccharomyces cerevisiae. We cloned an S. cerevisiae gene, YEA4, whose product is homologous to the Kluyveromyces lactis uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) transporter. An epitope-tagged Yea4p localized mainly in the 10,000 x g pellet (P2), suggesting endoplasmic reticulum (ER) localization. Membrane vesicles from the P2 fraction showed an 8-fold higher UDP-GlcNAc transport activity in cells harboring a multicopy YEA4 plasmid than in cells harboring vector alone. The activity distribution is identical with the protein distribution in P2, whether the gene is overexpressed or not, suggesting its native localization in P2. Immunolocalization of epitope-tagged Yea4p further revealed ER localization. The increase in transport activity due to the YEA4 overexpression is specific for UDP-GlcNAc, but not for UDP-galactose and GDP-mannose. Deltayea4-disrupted cells showed a reduced rate of UDP-GlcNAc transport, contained less chitin, and were larger and rounder in shape than the wild type cells. Our results indicate that YEA4 encodes an ER-localized UDP-GlcNAc transporter that is required for cell wall chitin synthesis in S. cerevisiae.

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.

HAS1 was expressed as a FLAG-tagged HAS1 fusion protein in COS-1 cells. This recombinant protein was extracted with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) from the membrane fraction and purified by anti-FLAG affinity chromatography and subsequent SDS-polyacrylamide gel electrophoresis. A protein solubilized from the one single band on the gel was able to synthesize hyaluronan when incubated with UDP-GlcNAc and UDP-GlcA as donor substrates without any further additions. The detergent-solubilized and purified HAS1 protein, however, exhibited quite different kinetic properties from the membrane-bound protein. When assayed under the reconstitutive conditions where the reaction mixture was layered onto the buffer containing high concentration of CHAPS, the activity was enhanced and the kinetic properties became similar to those of the membrane-bound protein. In addition, a HAS1 gene product by an in vitro transcription/translation system also showed HAS1 activity under the reconstitutive conditions. To our surprise, when incubated with UDP-GlcNAc alone, the protein was found to synthesize chito-oligosaccharide. Taking advantage of these enzyme reaction properties, active sites on the protein involved in for hyaluronan and chito-oligosaccharide synthesis were characterized. Site-directed mutagenesis induced in the cytoplasmic central loop domain of the protein revealed that several amino acid residues conserved among those domains of various proteins of a HAS family were essential for both hyaluronan and chito-oligosaccharide syntheses but one of them was not for chito-oligosaccharide synthesis. The substitutions that caused partial or severe loss of the activity gave no significant changes of the K(m) values of the mutated proteins, suggesting that no conformational or other indirect changes were involved in the effect. Taken together, the results suggest that the HAS1 protein alone is able to synthesize hyaluronan and different amino acid residues on the cytoplasmic central loop domain are involved in transferring GlcNAc and GlcA residues, respectively.

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.

The Saccharomyces cerevisiae gene,YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion ofAGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1(glucosamine-6-phosphateacetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 enzymes was severely diminished. Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased K m value for acetyl-CoA, suggesting that the Phe142 and Tyr143 residues are essential for the catalysis.

Cell-surface glycoprotein receptors have varying numbers of N-glycan sites. In this issue of Cell, Lau et al. (2007) report that increasing intracellular UDP-GlcNAc leads to increased branching of N-glycans, increased receptor association with cell-surface galectin-3, and enhanced signaling. They also show that the kinetics of this response differ between growth-promoting receptors, which have 8-16 N-glycans, and those that induce growth arrest, which have very few N-glycans, suggesting that hexosamine flux may regulate the transition from growth to arrest.

The polysaccharide intercellular adhesin (PIA) is an important factor in the colonization of medical devices by Staphylococcus epidermidis. The genes encoding PIA production are organized in the icaADBC (intercellular adhesion) operon. To study the function of the individual genes, we have established an in vitro assay with UDP-N-acetylglucosamine, the substrate for PIA biosynthesis, and analyzed the products by thin-layer chromatography and mass spectrometry. IcaA alone exhibited a low N-acetylglucosaminyltransferase activity and represents the catalytic enzyme. Coexpression of icaA with icaD led to a significant increase in activity. The newly identified icaD gene is located between icaA and icaB and overlaps both genes. N-Acetylglucosamine oligomers produced by IcaAD reached a maximal length of 20 residues. Only when icaA and icaD were expressed together with icaC were oligomer chains that react with PIA-specific antiserum synthesized. IcaA and IcaD are located in the cytoplasmic membrane, and IcaC also has all the structural features of an integral membrane protein. These results indicate a close interaction between IcaA, IcaD, and IcaC. Tunicamycin and bacitracin did not affect the in vitro synthesis of PIA intermediates or the complete PIA biosynthesis in vivo, suggesting that a undecaprenyl phosphate carrier is not involved. IcaAD represents a novel protein combination among beta-glycosyltransferases.

In this study, we have characterized the activity of a uridine diphospho-N -acetylglucosamine:polypeptide-α-N- acetylglucosaminyltransferase (O -α-GlcNAc-transferase) from Trypanosoma cruzi . The activity is present in microsomal membranes and is responsible for the addition of O -linked α-N -acetylglucosamine to cell surface proteins. This preparation adds N- acetylglucosamine to a synthetic peptide KPPTTTTTTTTKPP containing the consensus threonine-rich dodecapeptide encoded by T. cruzi MUC gene (Di Noia, J. M., Sanchez D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146–24149). Incorporation ofN -[3H]acetylglucosamine is linearly dependent on incubation time and concentration of enzyme and substrate. The transferase activity has an optimal pH of 7.5- 8.5, requires Mn2+, is unaffected by tunicamycin or amphomycin, and is strongly inhibited by UDP. The optimized synthetic peptide acceptor for the cytosolic O -GlcNAc-transferase (YSDSPSTST) (Haltiwanger, R. S., Holt, G. D., and Hart, G. W. (1990)J. Biol. Chem. 265, 2563–2568) is not a substrate for this enzyme. The glycosylated KPPTTTTTTTTKPP product is susceptible to base-catalyzed β-elimination, and the presence ofN- acetylglucosamine α-linked to threonine is supported by enzymatic digestion and nuclear magnetic resonance data. These results describe a unique biosynthetic pathway for T. cruzi surface mucin-like molecules, with potential chemotherapeutic implications.

Background UDP- N -acetylglucosamine enolpyruvyl transferase (MurA), catalyses the first committed step of bacterial cell wall biosynthesis and is a target for the antibiotic fosfomycin. The only other known enolpyruvyl transferase is 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme involved in the shikimic acid pathway and the target for the herbicide glyphosate. Inhibitors of enolypyruvyl transferases are of biotechnological interest as MurA and EPSP synthase are found exclusively in plants and microbes. Results The crystal structure of Escherichia coli MurA complexed with UDP- N -acetylglucosamine (UDP-GlcNAc) and fosfomycin has been determined at 1.8 a resolution. The structure consists of two domains with the active site located between them. The domains have a very similar secondary structure, and the overall protein architecture is similar to that of EPSP synthase. The fosfomycin molecule is covalently bound to the cysteine residue Cys115, whereas UDP-GlcNAc makes several hydrogen-bonding interactions with residues from both domains. Conclusions The present structure reveals the mode of binding of the natural substrate UDP-GlcNAc and of the drug fosfomycin, and provides information on the residues involved in catalysis. These results should aid the design of inhibitors which would interfere with enzyme-catalyzed reactions in the early stage of the bacterial cell wall biosynthesis. Furthermore, the crystal structure of MurA provides a model for predicting active-site residues in EPSP synthase that may be involved in catalysis and substrate binding.