Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.

The unfolded protein response has a protective role in yeast models of classic galactosemia.

Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.

Optimised chemical synthesis of 5-substituted UDP-sugars and their evaluation as glycosyltransferase inhibitors.

We have investigated the applicability of different chemical methods for pyrophosphate bond formation to the synthesis of 5-substituted UDP-galactose and UDP-N-acetylglucosamine derivatives. The use of phosphoromorpholidate chemistry, in conjunction with N-methyl imidazolium chloride as the promoter, was identified as the most reliable synthetic protocol for the preparation of these non-natural sugar-nucleotides. Under these conditions, the primary synthetic targets 5-iodo UDP-galactose and 5-iodo UDP-N-acetylglucosamine were consistently obtained in isolated yields of 40-43%. Both 5-iodo UDP-sugars were used successfully as substrates in the Suzuki-Miyaura cross-coupling with 5-formylthien-2-ylboronic acid under aqueous conditions. Importantly, 5-iodo UDP-GlcNAc and 5-(5-formylthien-2-yl) UDP-GlcNAc showed moderate inhibitory activity against the GlcNAc transferase GnT-V, providing the first examples for the inhibition of a GlcNAc transferase by a base-modified donor analogue.

A highly efficient galactokinase from Bifidobacterium infantis with broad substrate specificity.

Galactokinase (GalK), particularly GalK from Escherichia coli, has been widely employed for the synthesis of sugar-1-phosphates. In this study, a GalK from Bifidobacterium infantis ATCC 15697 (BiGalK) was cloned and over-expressed with a yield of over 80 mg/L cell cultures. The k(cat)/K(m) value of recombinant BiGalK toward galactose (164 s(-1) mM(-1)) is 296 times higher than that of GalK from E. coli, indicating that BiGalK is much more efficient in the phosphorylation of galactose. The enzyme also exhibits activity toward galacturonic acid, which has never been observed on other wild type GalKs. Further activity assays showed that BiGalK has broad substrate specificity toward both sugars and phosphate donors. These features make BiGalK an attractive candidate for the large scale preparation of galactose-1-phosphate and derivatives.

[Simple synthesis of P¹-(11-phenoxyundecyl)-P²-(2-acetamido-2-deoxy-α-D-galactopyranosyl) diphosphate].

A simple scheme of synthesis of P¹-(11-phenoxyundecyl)-P²-(2-acetamido-2-deoxy-α-D-galactopyranosyl) diphosphate synthetic lipid acceptor for glycosyltransferases participating in gram-negative bacteria O-antigenic polysaccharides is suggested.

A pulsed amperometric detection method of galactose 1-phosphate for galactosemia diagnosis.

Galactose 1-phosphate uridyltransferase deficiency causes the accumulation of galactose and galactose 1-phosphate (Gal 1-P) in the blood. We describe a new pulsed amperometric detection method for determining Gal 1-P levels as a pathognomic marker for the diagnosis of galactosemia. The method uses high-performance anion-exchange chromatography with pulsed amperometric detection. In an anion-exchange column, the analytes were separated in 5 min by the eluent mixture of 40 mM NaOH and 40 mM Na(2)CO(3). The detection limit (signal to noise ratio of 3) to Gal 1-P was 30 microg/dL. The linear dynamic range was 3.0-50 mg/dL (r(2)=0.9999). The mean recoveries of Gal 1-P for intra- and interday assays were 97.55-103.78%. This method clearly separated the type I galactosemia patients from the normal group and is a practical procedure for the rapid diagnosis of galactosemia.

Carbohydrate ligands of human C-reactive protein: binding of neoglycoproteins containing galactose-6-phosphate and galactose-terminated disaccharide.

Binding of carbohydrate ligand by human C-reactive protein (CRP), in both native form and structurally deviated form (neoCRP or mCRP), was investigated using galactose-6-phosphate (Gal6P)- and Galbeta3GalNAc-containing bovine serum albumin (BSA) derivatives. To this end, we synthesized glycosides of Gal6P and Galbeta3GalNAc that can potentially generate a terminal aldehyde group. omega-Aldehydo glycosides were then conjugated to BSA via reductive amination. Using these neoglycoproteins, we showed that: (1) Gal6P-BSA and Galbeta3GalNAc-BSA bound to both forms of CRP, the former with or without calcium and the latter only in the absence of calcium; (2) phosphate-containing ligands can be bound with or without calcium, but the binding is much stronger in the presence of calcium than in the absence, underscoring the importance of direct coordination of phosphate to two calcium ions observed in the X-ray structure of phosphorylcholine (PC)-CRP complex; (3) cross-inhibition studies further corroborated the hypothesis that binding sites of PC and sugar are contiguous; (4) while PC-BSA bound to the native CRP over a wide pH range of 4.5 to 9, all the carbohydrate ligands and protamine-BSA (poly-cation-based ligand) exhibited optimal binding at around pH 6 to 6.5; and (5) ligand-binding conformation of mCRP appears to be more fragile than that of the native CRP in the acidic media (pH < 6).

Chemical confirmation of a pentavalent phosphorane in complex with beta-phosphoglucomutase.

This communication reports the X-ray crystal structure of the alpha-d-galactose-1-phosphate complex with that of Lactococcus lactis beta-phosphoglucomutase (beta-PGM) crystallized in the presence of Mg2+ cofactor and the enzyme-to-phosphorus ratio determined by protein and phosphate analyses of the crystalline complex. The 1:1 ratio determined for this complex was compared to the 1:2 ratio determined for the crystals of beta-PGM grown in the presence of substrate and Mg2+ cofactor. This result verifies the published structure assignment of this latter complex as the phosphorane adduct formed by covalent bonding between the active site Asp8 carboxylate to the C(1)phosphorus of the beta-glucose 1,6-bisphosphate ligand and rules out the proposal of a beta-PGM-glucose-6-phosphate-1-MgF3- complex.

Protective effect of L-cysteine and glutathione on the modulated suckling rat brain Na+, K+, -ATPase and Mg2+ -ATPase activities induced by the in vitro galactosaemia.

UNLABELLED: Galactosaemia is an inborn error of galactose (Gal) metabolism characterized by irreversible brain damage. The aim of this study was to evaluate whether the antioxidants L-cysteine (Cys) and the reduced glutathione (GSH) could reverse the alterations of brain total antioxidant status (TAS) and the modulated activities of the enzymes Na+,K+ -ATPase and Mg2+ -ATPase in in vitro galactosaemia. Mixture A (mix. A: galactose-1-phosphate (Gal-1-P, 2mM) plus galactitol (Galtol, 2mM) plus Gal (4mM) = classical galactosaemia) or Mixture B (mix. B: Galtol (2mM) plus Gal (1mM) = galactokinase deficiency galactosaemia) were preincubated in the presence or absence of Cys (0.83mM) or GSH (0.83 mM) with whole brain homogenates of suckling rats at 37 degrees C for 1h. TAS and the enzyme activities were determined spectrophotometrically. The preincubation of brain homogenates with mix. A or mix. B resulted in a decrease of TAS to 30% (P < 0.01), while the presence of Cys or GSH increased TAS to 20% (P < 0.01) and 60% ( P < 0.001), respectively. The antioxidants reversed the inhibited Na+,K+ -ATPase by mix. A or mix. B and the stimulated Mg2+ -ATPase by mix. B to control values, whereas no effect was observed on the enormously activated Mg2+ -ATPase by mix. A. CONCLUSIONS: (a) Gal and its derivatives may produce free radicals in the suckling rat brain, reported for first time, (b) Na+,K+ -ATPase inhibition and Mg2+ -ATPase activation are probably due to the oxidative stress from the above compounds, (c) Cys or GSH could play a protective role reversing the inhibited Na+,K+ -ATPase toward normal in in vitro galactosaemia and (d) the addition of the above antioxidants may reduce the consequences of brain Mg2+ -ATPase activation by Gal and Galtol in galactokinase deficiency galactosaemia.

Conformation of a rigid tetrasaccharide epitope in the capsular polysaccharide of Vibrio cholerae O139.

A newly reported strain of Vibrio cholerae, known as strain O139 Bengal, is the first instance of an encapsulated strain that has caused epidemic cholera. The O-antigenic capsule is the critical antigen for protective immunity. Since mapping of the antigenic epitopes will assist in the development of a protein conjugate vaccine based on the capsular polysaccharide, we have undertaken a study of the three-dimensional conformation of the polysaccharide. It contains six residues in the repeating subunit with the unusual feature of a 4,6 cyclic phosphate on a beta-galactopyranoside. A structural epitope composed of four of the residues is somewhat similar to the Lewis(b) blood group tetrasaccharide. Polysaccharide samples enriched in (13)C have been prepared by growth of the bacteria in (13)C-enriched medium. Multidimensional heteronuclear NMR and molecular modeling studies are reported, which show that the O139 tetrasaccharide adopts a compact and tightly folded conformation that is relatively rigid and similar to the Le(b) conformation. The cyclic phosphate on the beta-galactopyranoside residue is in contact with the colitose residue linked to the beta-GlcNAc.

Beta-1,3-galactosyl-N-acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082: characterization, partial purification and relation to mucin degradation.

A new enzyme has been characterized in a cell-free extract of Bifidobacterium bifidum that catalysed the reversible phosphorolytic cleavage of beta-1,3-galacto-oligosaccharides. In the presence of Pi, the phosphorolysis reaction was favoured and was accompanied by a Walden reaction. Cleavage of the beta-glycosidic linkage gave an alpha-galactoside derivative (alpha-D-galactose 1-phosphate). The enzyme possesses a high specificity for beta-D-galactosido-(1, 3)-N-acetylglucosamine and beta-D-galactosido-(1, 3)-N-acetylgalactosamine. This purified intracellular enzyme had an estimated molecular mass of 140 kDa. The galactophosphorolytic activity on disaccharides was optimal at pH 6-6.5 and the reverse reaction was optimal at pH 5.5-6. The temperature optimum for phosphorolysis and the reverse reaction was approx. 50-55 degrees C. This enzyme is of particular interest in degrading some beta-D-Gal(1, 3) linkages and should be classified as EC 2.4.1.-.

Structure of the O-antigen of Vibrio cholerae O155 that shares a putative D-galactose 4,6-cyclophosphate-associated epitope with V. cholerae O139 Bengal.

The O-specific polysaccharide of Vibrio cholerae 0155 was studied by sugar and methylation analyses, dephosphorylation with 48% hydrofluoric acid, 1H- and 13C-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and heteronuclear single-quantum coherence (HSQC) experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established: carbohydrate sequence [see text]. An unusual component, D-galactose 4,6-cyclophosphate, has been reported previously as a component of the capsular polysaccharide and O-antigen of V. cholerae O139 Bengal and appears to be responsible for the known serological cross-reactivity between V. cholerae O139 and O155.

Crystal structures and mechanism of 6-phospho-beta-galactosidase from Lactococcus lactis.

The initial structural model of 6-phospho-beta-galactosidase from Lactococcus lactis was refined to an R-factor of 16.4% (R[free] = 23.6%) to 2.3 A resolution (1 A = 0.1 nm), and the structures of three other crystal forms were solved by molecular replacement. The four structural models are essentially identical. The catalytic center of the enzyme is approximately at the mass center of the molecule and can only be reached through a 20 A long channel, which is observed with an "open" or "closed" entrance. The closed entrance is probably too small for the educt lactose-6-phosphate to enter, but large enough for the first product glucose to leave. Among the presented structures is a complex between an almost inactive mutant and the second product galactose-6-phosphate, which is exclusively bound at side-chains. A superposition (onto the native enzyme) of galactose-6-phosphate as bound to the mutant suggests the geometry of a postulated covalent intermediate. The binding mode of the educt was modeled, starting from the bound galactose-6-phosphate. A tightly fixed tryptophan is used as a chopping-board for splitting the disaccharide, and several other aromatic residues in the active center cavity are likely to participate in substrate transport/binding.

Structure of the capsular polysaccharide of Vibrio cholerae O139 synonym Bengal containing D-galactose 4,6-cyclophosphate.

The capsular polysaccharide (CPS) of Vibrio cholerae O139 synonym Bengal, which is thought to carry determinants of O-specificity, was isolated by phenol/water extraction followed by delipidation of the contaminating lipopolysaccharide at pH 4.2 and gel-permeation chromatography. The CPS contained D-galactose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), D-galacturonic acid (D-GalA), and phosphate. The CPS was studied by NMR spectroscopy, methylation analysis, and selective degradations, including partial acid hydrolysis at pH 3.1 and dephosphorylation with aqueous 48% hydrofluoric acid, which both resulted in complete cleavage of Col. It was concluded that the CPS is built up of hexasaccharide repeating units containing inter alia D-galactose 4,6-cyclophosphate and having the following structure [structure: see text] These data basically confirm the structure of the V. cholerae CPS proposed on the basis of an NMR study [L. M. Preston et al. (1995) J. Bacteriol. 177, 835-838] and specify exactly the absolute configurations of the constituent monosaccharides and the position of the cyclic phosphate.

Lipid A and related compounds. XXVII. An efficient synthesis of D-galactosamine-4-phosphate analogs of lipid A via a novel key intermediate.

A new methodology for chemical differentiation of one amino and four hydroxyl groups of D-galactosamine derivatives and its application for the synthesis of D-galactosamine-4-phosphate analogs of lipid A are described. Preliminary examination of biological activity revealed that the synthetic monosaccharides show mitogenic activity.

Structure of the extracellular polysaccharide from slime-forming Lactococcus lactis subsp. cremoris SBT 0495.

The extracellular polysaccharide obtained from slime-forming Lactococcus lactis subsp. cremoris SBT 0495 is composed of D-glucose, D-galactose, L-rhamnose, and phosphate. Methylation analysis of the native and dephosphorylated polysaccharides provided information on the linkage of the sugar residues and the location of the phosphate group. N.m.r. spectroscopy confirmed the structure of the polysaccharide, which is assigned the following repeating-unit: [formula: see text]

[Biopolymer fragments containing residues of glycosylphosphtates. 6. Synthesis of phosphodiethers of alpha-D-galactopyranose--fragments of yeast phosphogalactanes from Sporobolomyces and capsule antigen of Escherichia coli K52]. / Fragmenty biopolimerov, soderzhashchikh ostatki glikozilfosfatov. 6. Sintez fosfodiéfirov alpha-D-galaktopiranozy--fragmentov drozhzhevykh fosfogalaktanov Sporobolomyces i kapsul'nogo antigena Escherichia coli K52.

Syntheses of methyl 6-(alpha-D-galactopyranosyl phosphate)- and p-nitrophenyl 3-(alpha-D-galactopyranosyl phosphate)-alpha-D-galactopyranosides were performed through coupling of 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl hydrogenphosphonate with the partially acylated galactopyranosides, in the presence of trimethylacetyl chloride, followed by oxidation and debenzoylation. The data of 1H, 13C, and 31P NMR spectra of the synthesized phosphate diesters are reported.