Biochemical characterization of an α1,2-colitosyltransferase from Escherichia coli O55:H7.

Colitose, also known as 3,6-dideoxy-L-galactose or 3-deoxy-L-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an L-fucokinase/GDP-L-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coliO55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Galß1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (kcat/Km= 9.2 min(-1)mM(-1)) as that toward GDP-colitose (kcat/Km= 12 min(-1)mM(-1)). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.

Enzymatic and regulatory properties of the trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum.

Trehalose-6-phosphate synthase plays an important role in trehalose metabolism. It catalyzes the transfer of glucose from UDP-glucose (UDPG) to glucose 6-phosphate to produce trehalose-6-phosphate. Herein we describe the characterization of a trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum. The dimeric enzyme could utilize UDPG, ADP-Glucose (ADPG) and GDP-Glucose (GDPG) as glycosyl donors and various phosphorylated monosaccharides as glycosyl acceptors. The optimal temperature and pH were found to be 60 °C and pH 6, and the enzyme exhibited notable pH and thermal stability. The enzymatic activity could be stimulated by divalent metal ions and polyanions heparin and chondroitin sulfate. Moreover, the protein was considerably resistant to additives ethanol, EDTA, urea, DTT, SDS, ß-mercaptoethanol, methanol, isopropanol and n-butanol. Molecular modeling and mutagenesis analysis revealed that the N-loop region was important for the catalytic efficiency of the enzyme, indicating different roles of N-loop sequences in different trehalose-6-phosphate synthases.

A convenient synthesis of GDP-D-rhamnose: the donor substrate for D-rhamnosyltransferase WbpZ from Pseudomonas aeruginosa PAO1.

Gram negative bacteria have lipopolysaccharides (LPS) that are critical for their survival. LPS molecules are composed of antigenic exopolysaccharide chains (O antigens). We are interested in discovering the enzymes involved in the biosynthesis of O antigens in Pseudomonas aeruginosa. The common polysaccharide antigen contains α-linked D-rhamnose residues. We have now synthesized GDP-D-rhamnose by a convenient synthesis in aqueous solution, and have shown that it can be used without extensive purification as the donor substrate for D-rhamnosyltransferase (WbpZ) from the P. aeruginosa strain PAO1. The availability of this nucleotide sugar preparation allows for characterization of D-rhamnosyltransferases.

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-ß-D-virenose biosynthesis.

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars ß-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-ß-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-ß-D-virenose and LPS structure in C. burnetii.

Accommodation of GDP-linked sugars in the active site of GDP-perosamine synthase.

Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.

A fast synthetic route to GDP-sugars modified at the nucleobase.

The direct structural modification of GDP-mannose via the bromination and Suzuki-Miyaura cross-coupling of the unprotected sugar-nucleotide, to produce 8-substituted fluorescent analogues of GDP-mannose.

Stereoselective chemical synthesis of sugar nucleotides via direct displacement of acylated glycosyl bromides.

[structure: see text]. The use of Leloir glycosyltransferases to prepare biologically relevant oligosaccharides and glycoconjugates requires access to sugar nucleoside diphosphates, which are notoriously difficult to efficiently synthesize and purify. We report a novel stereoselective route to UDP- and GDP-alpha-D-mannose as well as UDP- and GDP-beta-L-fucose via direct displacement of acylated glycosyl bromides with nucleoside 5'-diphosphates.

Structure and function of GDP-mannose-3',5'-epimerase: an enzyme which performs three chemical reactions at the same active site.

GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana catalyzes the epimerization of both the 3' and 5' positions of GDP-alpha-D-mannose to yield GDP-beta-L-galactose. Production of the C5' epimer of GDP-alpha-D-mannose, GDP-beta-L-gulose, has also been reported. The reaction occurs as part of vitamin C biosynthesis in plants. We have determined structures of complexes of GME with GDP-alpha-D-mannose, GDP-beta-L-galactose, and a mixture of GDP-beta-L-gulose with GDP-beta-L-4-keto-gulose to resolutions varying from 2.0 to 1.4 A. The enzyme has the classical extended short-chain dehydratase/reductase (SDR) fold. We have confirmed that GME establishes an equilibrium between two products, GDP-beta-L-galactose and GDP-beta-L-gulose. The reaction proceeds by C4' oxidation of GDP-alpha-D-mannose followed by epimerization of the C5' position to give GDP-beta-L-4-keto-gulose. This intermediate is either reduced to give GDP-beta-L-gulose or the C3' position is epimerized to give GDP-beta-L-4-keto-galactose, then C4' is reduced to GDP-beta-L-galactose. The combination of oxidation, epimerization, and reduction in a single active site is unusual. Structural analysis coupled to site-directed mutagenesis suggests C145 and K217 as the acid/base pair responsible for both epimerizations. On the basis of the structure of the GDP-beta-L-gulose/GDP-beta-L-4-keto-gulose co-complex, we predict that a ring flip occurs during the first epimerization and that a boat intermediate is likely for the second epimerization. Comparison of GME with other SDR enzymes known to abstract a protein alpha to the keto function of a carbohydrate identifies key common features.

GDP-mannose 3',5'-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants.

Despite its importance for agriculture, bioindustry, and nutrition, the fundamental process of L-ascorbic acid (vitamin C) biosynthesis in plants is not completely elucidated, and little is known about its regulation. The recently identified GDP-Man 3',5'-epimerase catalyzes a reversible epimerization of GDP-D-mannose that precedes the committed step in the biosynthesis of vitamin C, resulting in the hydrolysis of the highly energetic glycosyl-pyrophosphoryl linkage. Here, we characterize the native and recombinant GDP-Man 3',5'-epimerase of Arabidopsis thaliana. GDP and GDP-D-glucose are potent competitive inhibitors of the enzyme, whereas GDP-L-fucose gives a complex type of inhibition. The epimerase contains a modified version of the NAD binding motif and is inhibited by NAD(P)H and stimulated by NAD(P)+. A feedback inhibition of vitamin C biosynthesis is observed apparently at the level of GDP-Man 3',5'-epimerase. The epimerase catalyzes at least two distinct epimerization reactions and releases, besides the well known GDP-l-galactose, a novel intermediate: GDP-L-gulose. The yield of the epimerization varies and seems to depend on the molecular form of the enzyme. Both recombinant and native enzymes co-purified with a Hsp70 heat-shock protein (Escherichia coli DnaK and A. thaliana Hsc70.3, respectively). We speculate, therefore, that the Hsp70 molecular chaperones might be involved in folding and/or regulation of the epimerase. In summary, the plant epimerase undergoes a complex regulation and could control the carbon flux into the vitamin C pathway in response to the redox state of the cell, stress conditions, and GDP-sugar demand for the cell wall/glycoprotein biosynthesis. Exogenous L-gulose and L-gulono-1,4-lactone serve as direct precursors of l-ascorbic acid in plant cells. We propose an L-gulose pathway for the de novo biosynthesis of vitamin C in plants.

Structure of the MUR1 GDP-mannose 4,6-dehydratase from Arabidopsis thaliana: implications for ligand binding and specificity.

GDP-D-mannose 4,6-dehydratase catalyzes the first step in the de novo synthesis of GDP-L-fucose, the activated form of L-fucose, which is a component of glycoconjugates in plants known to be important to the development and strength of stem tissues. We have determined the three-dimensional structure of the MUR1 dehydratase isoform from Arabidopsis thaliana complexed with its NADPH cofactor as well as with the ligands GDP and GDP-D-rhamnose. MUR1 is a member of the nucleoside-diphosphosugar modifying subclass of the short-chain dehydrogenase/reductase enzyme family, having homologous structures and a conserved catalytic triad of Lys, Tyr, and Ser/Thr residues. MUR1 is the first member of this subfamily to be observed as a tetramer, the interface of which reveals a close and intimate overlap of neighboring NADP(+)-binding sites. The GDP moiety of the substrate also binds in an unusual syn conformation. The protein-ligand interactions around the hexose moiety of the substrate support the importance of the conserved triad residues and an additional Glu side chain serving as a general base for catalysis. Phe and Arg side chains close to the hexose ring may serve to confer substrate specificity at the O2 position. In the MUR1/GDP-D-rhamnose complex, a single unique monomer within the protein tetramer that has an unoccupied substrate site highlights the conformational changes that accompany substrate binding and may suggest the existence of negative cooperativity in MUR1 function.

A high-performance liquid chromatography radio method for determination of L-ascorbic acid and guanosine 5'-diphosphate-l-galactose, key metabolites of the plant vitamin C pathway.

A simple, rapid, and quantitative high-pressure liquid chromatography radio method is described for the determination of in vivo (14)C-labeled l-ascorbate, dehydro-l-ascorbate, and total l-ascorbate of Arabidopsis thaliana cell suspensions upon incubation of cultures with exogenous d-[(14)C]mannose. The same radio-HPLC conditions can be used to follow the products of in vitro enzymatic conversions of GDP-d-mannose by enzyme extracts of A. thaliana, namely GDP-l-galactose, GDP-4"-keto,6"-deoxy-d-mannose, and GDP-l-fucose. In particular, an accurate assay for GDP-d-mannose 3",5"-epimerase, a key enzyme of the plant vitamin C pathway, is presented.

Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose.

The glycan chain repeats of the S-layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 contain d-glycero-d-manno-heptose, which has also been described as constituent of lipopolysaccharide cores of Gram-negative bacteria. The four genes required for biosynthesis of the nucleotide-activated form GDP-d-glycero-d-manno-heptose were cloned, sequenced, and overexpressed in Escherichia coli, and the corresponding enzymes GmhA, GmhB, GmhC, and GmhD were purified to homogeneity. The isomerase GmhA catalyzed the conversion of d-sedoheptulose 7-phosphate to d-glycero-d-manno-heptose 7-phosphate, and the phosphokinase GmhB added a phosphate group to form d-glycero-d-manno-heptose 1,7-bisphosphate. The phosphatase GmhC removed the phosphate in the C-7 position, and the intermediate d-glycero-alpha-d-manno-heptose 1-phosphate was eventually activated with GTP by the pyrophosphorylase GmhD to yield the final product GDP-d-glycero-alpha-d-manno-heptose. The intermediate and end products were analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to confirm the structure of these substances. This is the first report of the biosynthesis of GDP-d-glycero-alpha-d-manno-heptose in Gram-positive organisms. In addition, we propose a pathway for biosynthesis of the nucleotide-activated form of l-glycero-d-manno-heptose.

Glucosylation of glycosylphosphatidylinositol membrane anchors: identification of uridine diphosphate-glucose as the direct donor for side chain modification in Toxoplasma gondii using carbohydrate analogues.

Toxoplasma gondii is an obligate intracellular parasite of the phylum apicomplexa and a common and often life-threatening opportunistic infection associated with AIDS. A family of parasite-specific glycosylphosphatidylinositols containing a novel glucosylated side chain has been shown to be highly immunogenic in humans (Striepen et al. (1997) J. Mol. Biol. 266, 797-813). In contrast to trypanosomes in T. gondii side chain modification takes place before addition to protein in the endoplasmic reticulum. The biosynthesis of these modifications was studied in an in vitro system prepared from hypotonically lysed T. gondii parasites. Radiolabeled glucose-containing glycosylphosphatidylinositol precursors were synthesized by T. gondii membrane preparations upon incubation with uridine diphosphate-[3H]glucose. Synthesis of glucosylated glycolipids took place only in the presence of exogenous uridine diphosphate-glucose and was stimulated by unlabeled uridine diphosphate-glucose in a dose-dependent manner. In contrast to glycosylphosphatidylinositol mannosylation, glucosylation was shown to be insensitive to amphomycin treatment. In addition, the glucose analogue 2-deoxy-D-glucose was used to trace the glycosylphosphatidylinositol glucosylation pathway. Detailed analysis of glycolipids synthesized in vitro in the presence of UDP and GDP derivatives of D-glucose and 2-deoxy-D-glucose ruled out an involvement of dolichol phosphate-glucose and demonstrates direct transfer of glucose from uridine diphosphate-glucose.

A high-yield, enzymatic synthesis of GDP-D-[3H]arabinose and GDP-L-[3H]fucose.

For assays involving glycosyltransferases or transporters, several GDP-sugars are either commercially unavailable or expensive. We describe an enzymatic synthesis of GDP-d-[3H]arabinosep and GDP-l-[3H]fucose that yields 66-95% nucleotide-sugar from the appropriate radiolabeled sugar in less than 30 min. The coupled reaction requires Mg2+, ATP, and GTP along with the appropriate radioactive monosaccharide, sugar-1-kinase, and pyrophosphorylase. The latter two activities are present in a cytosolic fraction of Crithidia fasciculata, which is easily grown at room temperature in simple culture medium without serum or added CO2. Addition of commercial yeast inorganic pyrophosphatase shifts the equilibrium of the pyrophosphorylase reaction toward nucleotide-sugar formation. To verify that these nucleotide-sugars are biologically active, we tested their ability to serve as substrates for glycosyltransferases. GDP-l-[3H]fucose functions as the donor substrate for recombinant human fucosyltransferase V, and GDP-d-[3H]arabinosep serves as the donor substrate for the arabinosyltransferase activities present in Leishmania major microsomes.

The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries.

Human recombinant CD38 catalyzes the formation of both cyclic ADP-ribose and ADP-ribose products from NAD+ and hydrolyzes cyclic ADP-ribose to ADP-ribose. The corresponding GDP products are formed from NGD+. The enzyme was characterized by substrate and inhibition kinetics, exchange studies, rapid-quench reactions, and stopped-flow-fluorescence spectroscopy to establish the reaction mechanism and energetics for individual steps. Noncyclizable substrates NMN+ and nicotinamide-7-deaza-hypoxanthine dinucleotide (7-deaza NHD+) were rapidly hydrolyzed by the enzyme. The kcat for NMN+ was 5-fold higher than that of NAD+ and has the greatest reported kcat of any substrate for CD38. 7-deaza-NHD+ was hydrolyzed at approximately one-third the rate of NHD+ but does not form a cyclic product. These results establish that a cyclic intermediate is not required for substrate hydrolysis. The ratio of methanolysis to hydrolysis for cADPR and NAD+ catalyzed by CD38 increases linearly with MeOH concentration. Both reactions produce predominantly the beta-methoxy riboside compound, with a relative nucleophilicity of MeOH to H2O of 11. These results indicate the existence of a stabilized cationic intermediate for all observed chemistries in the active site of CD38. The partitioning of this intermediate between cyclization, hydrolysis, and nicotinamide-exchange unites the mechanisms of CD38 chemistries. Steady-state and pre-steady-state parameters for the partition and exchange mechanisms allowed full characterization of the reaction coordinate. Stopped-flow methods indicate a burst of cGDPR formation followed by the steady-state reaction rate. A lag phase, which was NGD+ concentration dependent, was also observed. The burst size indicates that the dimeric enzyme has a single catalytic site formed by two subunits. Pre-steady-state quench experiments did not detect covalent intermediates. Nicotinamide hydrolysis of NGD+ precedes cyclization and the chemical quench decomposes the enzyme-bound species to a mixture of cyclic and hydrolysis products. The time dependence of this ratio indicated that nicotinamide bond-breakage occurs 4 times faster than the conversion of the intermediate to products. Product release is the overall rate-limiting step for enzyme reaction with NGD+.

Characterization of GDP-alpha-D-arabinopyranose, the precursor of D-Arap in Leishmania major lipophosphoglycan.

Protozoan parasites of the genus Leishmania synthesize a complex lipophosphoglycan (LPG), which is the major cell surface macromolecule. The LPG from Leishmania major contains beta-D-Arap-terminating side chains that are involved in regulating the attachment of the parasite to the midgut epithelium of its insect vector. An arabinose sugar nucleotide donor was identified in soluble extracts of L. major promastigotes. This sugar nucleotide was biosynthetically labeled with D-[2-3H]Glc and with D-[5-3H]Ara. The labeled sugar nucleotide generated [3H]Ara and GDP after mild acid hydrolysis. The absolute configuration of the arabinose was determined after reduction and acylation with a pure enantiomer of Mosher's acid chloride. The pyranose ring configuration was inferred from the ability of GDP-Ara to form borate complexes, and the anomeric configuration was deduced from the results of mild base hydrolysis experiments. Taken together these data suggest that the sugar nucleotide has the structure GDP-alpha-D-Arap. This sugar nucleotide has not been previously described from natural sources and may be unique to trypanosomatid protozoan parasites. Ara-1-PO4 and GDP-Ara were the only soluble metabolites labeled with [3H]Ara, and pulse-chase experiment data are consistent with them being precursors of the arabinosyl residues of LPG.