Characterization and investigation of substrate specificity of the sugar aminotransferase WecE from E. coli K12

WecE gene, encoding a sugar aminotransferase (SAT), has been cloned from E. coli K12 and expressed in E. coli BL21 (DE3). The enzyme was purified and characterized. WecE used TDP-4-keto-6-deoxy-D-glucose (TDP-D-Glc4O) and L-glutamate as a good amino acceptor and donor, respectively, leading to the production of TDP-4-amino-4,6-dideoxy-D-galactose (TDP-Fuc4N), which was identified by NMR studies. WecE also showed a similar activity for TDP-4-keto 6-deoxy-D-mannose (TDP-D-Man4O), but no activity for GDP-4-keto-6-deoxy-D-mannose (GDP-D-Man4O), suggesting that the nucleotide moiety would become a key determinant to the substrate specificity of amine acceptor for the activity of the SAT. Multiple alignments showed that SATs have four highly conserved motifs located around the active site and could be divided into three subgroups (VIalpha, VIbeta, and VIgamma) that might be closely related with their substrate specificities.     

In vitro characterization of the enzymes involved in TDP-D-forosamine biosynthesis in the spinosyn pathway of Saccharopolyspora spinosa

Forosamine (4-dimethylamino)-2,3,4,6-tetradeoxy-beta-D-threo-hexopyranose) is a highly deoxygenated sugar component of several important natural products, including the potent yet environmentally benign insecticide spinosyns. To study D-forosamine biosynthesis, the five genes (spnO, N, Q, R, and S) from the spinosyn gene cluster thought to be involved in the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-D-forosamine were cloned and heterologously expressed, and the corresponding proteins were purified and their activities examined in vitro. Previous work demonstrated that SpnQ functions as a pyridoxamine 5'-monophosphate (PMP)-dependent 3-dehydrase which, in the presence of the cellular reductase pairs ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase, catalyzes C-3 deoxygenation of TDP-4-keto-2,6-dideoxy-D-glucose. It was also established that SpnR functions as a transaminase which converts the SpnQ product, TDP-4-keto-2,3,6-trideoxy-D-glucose, to TDP-4-amino-2,3,4,6-tetradeoxy-D-glucose. The results presented here provide a full account of the characterization of SpnR and SpnQ and reveal that SpnO and SpnN functions as a 2,3-dehydrase and a 3-ketoreductase, respectively. These two enzymes act sequentially to catalyze C-2 deoxygenation of TDP-4-keto-6-deoxy-D-glucose to form the SpnQ substrate, TDP-4-keto-2,6-dideoxy-D-glucose. Evidence has also been obtained to show that SpnS functions as the 4-dimethyltransferase that converts the SpnR product to TDP-D-forosamine. Thus, the biochemical functions of the five enzymes involved in TDP-D-forosamine formation have now been fully elucidated. The steady-state kinetic parameters for the SpnQ-catalyzed reaction have been determined, and the substrate specificities of SpnQ and SpnR have been explored. The implications of this work for natural product glycodiversification and comparative mechanistic analysis of SpnQ and related NDP-sugar 3-dehydrases E1 and ColD are discussed.     

Characterization of the TDP-D-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-D-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate

Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (D-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-D-ravidosamine biosynthetic enzymes were identified. Through the activities of the isolated enzymes in vitro, it is demonstrated that ravD, ravE, ravIM, ravAMT and ravNMT encode TDP-D-glucose synthase, TDP-4-keto-6-deoxy-D-glucose-4,6-dehydratase, TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase, TDP-3-keto-6-deoxy-D-galactose-3-aminotransferase, and TDP-3-amino-3,6-dideoxy-D-galactose-N,N-dimethyl-transferase, respectively. A protocol for a one-pot enzymatic synthesis of TDP-D-ravidosamine has been developed. The results presented here now set the stage to produce TDP-D-ravidosamine routinely for glycosylation studies.     

Quantification of sugars and sugar phosphates in Arabidopsis thaliana tissues using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry

This work reports the development and optimisation of a negative ion mode on-line LC-ESI-MS/MS method for the sensitive targeted analysis of the key glycolytic intermediates, sugars and sugar phosphates from plants, using a porous graphitic carbon (PGC) stationary phase and an MS compatible mobile phase. Using this newly developed method, separation and detection of a solution of standard compounds is achieved in less than 20min. Target metabolite compounds were identified in plant extracts from their characteristic retention times, and product ion spectra. This on-line PGC-ESI-MS/MS method shows good linearity over the concentration range 0-100microM, selectivity, short analysis time, and limits of detection of 0.1microM for disaccharides trehalose (Tre), sucrose (Suc), and maltose, and 1.5microM for hexose phosphates fructose-6-phosphate (Fru6P), glucose-1-phosphate (Glc1P), and glucose-6-phosphate (Glc6P), and phosphoenolpyruvate (PEP). This paper describes details of our method and its application to the simultaneous quantitative analysis of soluble sugars and sugar phosphates from Arabidopsis thaliana tissues. We have demonstrated the utility of our method for the analysis of biological samples by applying it to the simultaneous quantitation of changes in soluble sugars and sugar phosphates in A. thaliana Columbia-0 (Col-0) and its starchless phosphoglucomutase (pgm) mutant over a 12-h light/12-h dark growth cycle.     

Hydrophilic interaction chromatography/electrospray mass spectrometry analysis of carbohydrate-related metabolites from Arabidopsis thaliana leaf tissue

This work describes the development and application of an on-line liquid chromatography/mass spectrometry (LC/MS) method using hydrophilic interaction chromatography (HILIC) coupled to negative ion mode electrospray ionisation ion trap mass spectrometry (ESI-MS) for the analysis of highly polar carbohydrate-related metabolites commonly found in plants, ranging from reducing and non-reducing sugars and sugar alcohols to sugar phosphates. Using this method, separation and detection of a mixture of eight authentic standard compounds containing glucose (Glc), sucrose (Suc), raffinose, verbascose, mannitol, maltitol, glucose-6-phosphate (Glc6P) and trehalose-6-phosphate (Tre6P) were achieved in less than 15 min. The method is rapid, robust, selective, and sensitive, with limits of detection (LODs) ranging from 0.2 microM obtained for neutral sugars, to 1.0 microM obtained for sugar alcohols, and 2.0 microM obtained for negatively charged sugar phosphates. We have studied the negative ion collision-induced dissociation (CID) fragmentation behaviour of the non-reducing raffinose family oligosaccharides (RFOs) raffinose, stachyose, and verbascose. Mainly Bi and Ci glycosidic and Ai cross-ring structurally informative cleavages are observed. We have applied this HILIC/ESI-MS method for the analysis of Arabidopsis thaliana wild-type Columbia-0 (Col-0) and its starchless phosphoglucomutase mutant (pgm1) leaf extracts. The method was used to quantify Glc, Suc, raffinose, and Glc6P in A. thaliana extracts. Data obtained using this HILIC/ESI-MS method were compared with those obtained using a comparable porous graphitic carbon-based LC/ESI-MS method.     

Synthesis of GDP-5-thiosugars and their use as glycosyl donor substrates for glycosyltransferases

Two thiopyranoside analogues of GDP-sugars, GDP-5-thio-d-mannose (14) and GDP-5-thio-l-fucose (15), were synthesized. The syntheses included the phosphorylations of tetra-O-acetyl-5-thio-d-mannosyl bromide (4) and tri-O-benzoyl-l-fucosyl bromide (6) with silver dibenzyl phosphate, deprotection of the phosphate groups, and condensation of the deprotected phosphates with GMP-imidazolidate (13) in the presence of MgCl(2). These GDP-sugar analogues were found to be donor substrates for alpha(1,2)mannosyltransferase and alpha(1,3)fucosyltransferase, affording a 5-thiomannose-containing disaccharide (18) and a 5-thiofucose-containing trisaccharide (21), respectively. The conformation of the disaccharide analogue 18 was similar to that of its native counterpart by ROESY. These findings for GDP-5-thiosugars together with previous demonstrations of enzymatic transfer from UDP-5-thiosugars will allow the production of panels of oligosaccharide analogues with hydrolase-resistant properties.     

Analysis of cytokinin nucleotides in coconut (Cocos nucifera L.) water using capillary zone electrophoresis-tandem mass spectrometry after solid-phase extraction

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is described for the separation and determination of six cytokinin nucleotides in coconut water. The best CZE separation for the six cytokinin nucleotide standards was achieved using a 25 mM ammonium formate/formic acid buffer (pH 3.8) and 2% (v/v) methanol with an applied gradient separation voltage (25 kV for 32 min, and then a linear gradient to 30 kV in 5 min, finally 30 kV to the end of separation) in less than 60 min. MS/MS with multiple reaction monitoring (MRM) detection was carried out to obtain sufficient selectivity and sensitivity for the cytokinin nucleotides. The combined use of on-line sample stacking and CZE-MS/MS achieved limits of detection (LODs) in the range of 0.06-0.19 microM for the six cytokinin nucleotides at a signal-to-noise ratio of 3. Furthermore, a novel dual-step SPE procedure was developed for the pre-concentration and purification of cytokinin nucleotides using Oasis HLB and Oasis MAX cartridges. The recoveries of the cytokinin nucleotides after the dual-step SPE were in the range of 44-71%. The combination of off-line SPE, on-line sample stacking and CZE-MS/MS approach was successfully applied to screen for endogenous cytokinin nucleotides present in coconut water sample. trans-Zeatin riboside-5'-monophosphate (ZMP) was detected and quantified in coconut water by CZE-MS/MS after SPE and on-line sample stacking.     

Characterization of protein encoded by spnR from the spinosyn gene cluster of Saccharopolyspora spinosa: mechanistic implications for forosamine biosynthesis

d-Forosamine is a 4-N,N-(dimethylamino)-2,3,4,6-tetradeoxy-alpha-d-threo-hexopyranose found in spinosyn produced by Saccharopolyspora spinosa. Studies of spinosyn biosynthesis in S. spinosa led to the isolation of the entire biosynthetic gene cluster. Heterologous expression of spnR, one putative gene in forosamine biosynthesis, in E. coli and purification of the SpnR protein identified it as an aminotransferase catalyzing the conversion of the 4-keto-2,3,6-trideoxy sugar intermediate to the corresponding 4-amino sugar product. Identification of SpnR function relied on the use of a stable TMP-phosphonate sugar in place of TDP-sugar substrate to determine the function of SpnR. This strategy may find general applicability for designing probes to study enzymes which catalyze the transformation of labile deoxysugar intermediates.     

近红外光谱技术用于白砂糖质量的实时监测研究

采用国产近红外在线分析仪,结合漫反射光谱分析技术,对广西某糖厂的白砂糖质量进行了在线应用研究.讨论了影响建模的因素,并提出一些解决问题的方案.采用SupNIR-4000型长波近红外在线分析仪,直接对产品传送带上的白砂糖进行扫描获得光谱数据,运用偏最小二乘法对实验数据进行多元统计分析建立数学模型,并进行了预测.样品色值的预测值和实测值间的相关系数为0.885,标准校正偏差为4.300,色值预测偏差小于糖厂要求的±10;样品浊度预测值和实测值间的相关系数为0.909,标准校正偏差为2.308,浊度预测偏差小于糖厂要求的±4.结果显示,模型预测结果符合糖厂要求,表明在线近红外技术在白砂糖质量监控中具有较好的应用前景.     

Analysis of sugar phosphates in plants by ion chromatography on a titanium dioxide column with pulsed amperometric detection

This paper describes the development of a practical method for the analysis of sugar phosphates from the model higher plant Arabidopsis thaliana by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The extraction method of sugar phosphates from higher plants was first optimized for HPAEC-PAD analysis. In order to improve the resolution in HPAEC-PAD, a column packed with titanium dioxide resin was used. The titanium dioxide column was used as a trap-column for sugar phosphates and nucleotides, for the removal of sample matrices. Sample pretreatment was achieved in-line and automatically using a six-port valve placed after the injection valve.     

On-line immunoaffinity column-liquid chromatography-tandem mass spectrometry method for trace analysis of diuron in wastewater treatment plant effluent sample

An on-line immunoaffinity column with liquid chromatography/tandem mass spectrometry (IAC-LC-MS/MS) method for the determination of diuron in water matrices was described. This method used a sol-gel immunoaffinity column (20 mm x 4 mm I.D.) for on-line sample cleanup and enrichment, a monolithic analytical column (100 mm x 4.6 mm I.D.) for separation, and a triple quadrupole mass spectrometer for quantitation. The major challenges for the on-line set-up were discussed. The optimized on-line protocol was emphasized by the fact that low limit of quantitation (LOQ) of 1.0 ng/L was achieved with only 2.5-mL sample. In addition, a satisfactory accuracy ( approximately 90% of recovery) and precision (<6% of relative standard deviation) at 50 ng/L concentration were also obtained. Due to the ability of the sol-gel immunoaffinity column to eliminate matrix effect, the on-line IAC-LC-MS/MS analysis method can reliably determine diuron in wastewater treatment plant effluent sample.     

Synthesis of fluorescently labeled alkylated DNA adduct standards and separation by capillary electrophoresis

DNA adducts are regarded as individual internal dosimeters for the exposure to chemical carcinogens. To date, the most sensitive method for DNA adduct analysis is the radioactive 32P-postlabeling method, which allows the detection of one adduct in 10(10) unmodified nucleotides in microg amounts of DNA. However, this technique suffers from disadvantages such as working with radioactive phosphorus and time-consuming chromatographic separation procedures. In addition, the simultaneous detection of adducts from different classes of carcinogens in a DNA sample is difficult. In order to overcome these drawbacks, we are developing a new detection method, comprising fluorescence labeling of DNA adducts, capillary electrophoretic (CE) separation, and on-line detection by monitoring laser-induced fluorescence (LIF). So far, we have evaluated the separation power and the detection limit of CE with fluorescently labeled standard compounds such as unmodified nucleotides or alkylated thymidines. For this purpose, we developed a universal method for labeling 5'-OH-mononucleosid-3'-dicyanoethyl-phosphates with fluorescent dyes based on the phosphoramidite technology for DNA synthesis. The separation of N3-methylated, N3-, O2- and O4-butylated thymidines from the unmodified nucleotide within a few minutes recommends CE-LIF as a powerful method for DNA adduct analysis.     

A highly sensitive CE-UV method with dynamic coating of silica-fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

A new highly sensitive capillary electrophoresis (CE) method applying dynamic coating and on-line stacking for the monitoring of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and the screening of inhibitors was developed. NPP1 and NPP3 are membrane glycoproteins that catalyze the hydrolysis nucleotides, e.g. convert adenosine 5'-triphosphate to adenosine 5'-monophosphate (AMP) and pyrophosphate. Enzymatic reactions were performed and directly subjected to CE analysis. Since the enzymatic activity was low, standard methods were insufficient. The detection of nanomolar AMP and other nucleotides could be achieved by field-enhanced sample injection and the addition of polybrene to the running buffer. The polycationic polymer caused a dynamic coating of the silica-fused capillary, resulting in a reversed electroosmotic flow. The nucleotides migrated in the direction of the electroosmotic flow, whereas the positively charged polybrene molecules moved in the opposite direction, resulting in a narrow sample zone over a long injection time. Using this on-line sensitivity enhancement technique, a more than 70-fold enrichment was achieved for AMP (limit of detection, 46 nM) along with a short migration time (5 min) without compromising separation efficiency and peak shape. The optimized CE conditions were as follows: fused-silica capillary (30 cm effective lengthx75 mum), electrokinetic injection for 60 s, 50 mM phosphate buffer pH 6.5, 0.002% polybrene, constant current of -60 muA, UV detection at 210 nm, uridine 5'-monophosphate as the internal standard. The new method was used to study enzyme kinetics and inhibitors. It opens an easy way to determine the activities of slowly metabolizing enzymes such as NPPs, which are of considerable interest as novel drug targets.     

Carbohydrate analysis by high-performance anion-exchange chromatography with pulsed amperometric detection: the potential is still growing

This article reviews recent advances of carbohydrate analysis by high-performance anion-exchange chromatography with pulsed amperometric detection. Starting from the paper of Dennis C. Johnson [1] in which the great analytical promise of such a technique was anticipated, a multitude of exciting new research possibilities have recently emerged. The great attractiveness of high-performance anion-exchange chromatography is largely due to its compatibility with such a sensitive, selective and reliable detection method as pulsed amperometry. This very good match between liquid chromatography and electrochemical detection has allowed the determination of carbohydrates in a variety of complex matrices, for instance, foods, beverages, diary and biotechnological products, vegetal tissues, and also in the area of clinical diagnostics. For this reason, the introduction of HPAEC-PAD into regulated methods is becoming increasingly accepted. A comprehensive collection of applications to carbohydrates and samples of interest is given, with special focus on the separation of closely related sugar compounds using dilute alkaline eluents. Advances in pulsed potential waveforms are also discussed, and a comparison with other liquid chromatographic methods addressed. 2-keto-3-deoxy-D-glycero-D-galactonononic acid; KDO, 2-keto-3-deoxyoctulosonic acid; FOS, fructooligosaccharides; GF5, GF6, and GF7, oligofructans: Hib, Haemophilus influenzae type b; FAB, fast atom bombardment; ESI, electrospray ionization; MALDI-TOF, matrix assisted laser desorption ionization-time of flight.     

The assembly of lipid-linked oligosaccharides in plant and animal membranes.

Membrane preparations from growing regions of pea stems and actively-dividing mouse L-cells form lipid-linked saccharides from GDP-mannose and UDP-N-acetylglucosamine. These lipids have properties which are consistent with those of mono- and di-phosphoryl polyisoprenyl derivatives. In experiments using plant membranes, the monophosphoryl derivative labeled with GDP-(14C) mannose contains mannose only, while the diphosphoryl derivative labeled with the same nucleotide sugar is heterogeneous, containing oligosaccharides corresponding to mannosaccharides of 5, 7, and 9--12 residues. Only the diphosphoryl polyisoprenyl derivatives are labeled with UDP-(14C)glucosamine and these contain predominantly chitobiose and N-acetylglucosamine itself. Unlabeled GDP-mannose added after UDP-N-acetyl-(14C)glucosamine results in the formation of higher lipid-linked oligosaccharides which are apparently the same as those which are labeled with GDP-(14C)mannose alone. Incubation of the membranes with GDP-(14C)mannose in the presence of Mn2+, unlabeled UDP-glucose or unlabeled UDP-N-acetylglucosamine results in marked changes in the accumulation of both the polyisoprenyl monophosphoryl mannose and polyisoprenyl diphosphoryl oligosaccharides. Animal cell membranes synthesise lipid-linked oligosaccharides when incubated with UDP-N-acetylglucosamine and GDP-mannose. These oligosaccharides are similar in size to those synthesised by the plant membranes but their formation is more efficient. The potential roles of these compounds in glycoprotein biosynthesis in both plant and animal tissues is discussed.     

Polynuclear heterocyclic compounds

When 2, 3(CO)-benzoylene-4-phenyl-5-keto-7, 7-dimethyl-5, 6, 7, 8-tetrahydroquinoline is catalytically hydrogenated using Adams catalyst, the five-membered ring carbonyl group is reduced to an alcohol group. The acetate, urethane, monoxime, and hydrochloride of the reduction product are prepared. Their IR spectra are measured. When 2, 3 (CO)-benzoylene-4-phenyl-5-keto-7, 7-dimethyl-5, 6, 7, 8-tetrahydroquinoline is reduced by zinc dust and glacial acetic acid, both carbonyl groups are reduced to CH₂, and 2, 3 (CH₂)-benzylene-4-phenyl-5-keto-7, 7-dimethyl-5, 6, 7, 8-tetrahydroquinoline is formed.For Part XIX see [2].     

Gas chromatographic determination of (phosphorylated) 2-keto-3-deoxyoctonic acid, heptoses and glucosamine in bacterial lipopolysaccharides after treatment with hydrofluoric acid, methanolysis and trifluoroacetylation

Quantification of phosphorylated sugar constituents of lipopolysaccharides has been performed by the following sequence: dephosphorylation by treatment with hydrofluoric acid, cleavage to monomeric constituents by methanolysis and analysis of the released sugars by capillary gas chromatography. Lipopolysaccharides of Salmonella minnesota Rd1P+, Bordetella pertussis NIH 114 and Vibrio cholerae, NAG and 95R strains, were used as model substances. Comparison of the chromatographic data obtained from hydrofluoric acid-treated and untreated lipopolysaccharide preparations indicated that all lipopolysaccharides examined contained one moiety of glucosamine bound to phosphate in a stable linkage. 2-Keto-3-deoxyoctonic acid appeared phosphorylated to a variable extent. Lipopolysaccharides of the two V. cholerae strains contained one moiety of fully phosphorylated 2-keto-3-deoxyoctonic acid, whereas in that of S. minnesota Rd1P+ only one of the three moieties was phosphorylated. Lipopolysaccharide of B. pertussis had one moiety of 2-keto-3-deoxyoctonic acid, ca. 70% phosphorylated. All four of the preparations examined contained L-glycero-D-manno-heptose in amounts varying from 2.6 to 5.2 moieties. In the lipopolysaccharides of B. pertussis and strain 95R of V. cholerae this sugar was unphosphorylated, whereas the two remaining strains contained one phosphorylated moiety of this sugar. Phosphorylated lipopolysaccharide constituents can be analysed by this approach on a 50-100 micrograms scale.     

Characterization of Nucleotides and Nucleotide Sugars in Candida albicans by High Performance Liquid Chromatography–Mass Spectrometry with a Porous Graphite Carbon Column

An optimized high performance liquid chromatography electrospray ionization/tandem mass spectrometry method was developed and optimized for the determination of seven nucleotides and nucleotide sugars in the extract of Candida albicans. The chromatographic separation was performed on a porous graphitic carbon column with gradient elution using acetonitrile and aqueous ammonia acetate containing 0.1% formic acid adjusted to pH 8.8. Negative-ion mode electrospray ionization mass spectrometry was applied to improve the selectivity and sensitivity of the analysis. The calibration curves showed excellent linearity over approximately three orders of magnitude with correlation coefficients exceeding 0.9946. The relative standard deviations of measurements were smaller than 4% (n = 5) and the accuracy of the intra-day and inter-day measurements were between 95.2% and 106.7%. The extraction recovery of each component was within satisfactory ranges. The validated method was applied to characterize the nucleotides metabolic profiles of the biofilm compared to the planktonic mode to evaluate changes of energy states in different growth modes of C. albicans. The levels of nucleotides and nucleotide sugar in the biofilm model treated with 5-fluoropyrimidine, baicalein, and sodium houttuyfonate were determined to investigate their effects on nucleotide metabolism and the mechanisms of antifungal agents.     

Gas chromatographic separation and mass spectrometric identification of mixtures of oxyphytosterol and oxycholesterol derivatives application to a phytosterol-enriched food

Pure individual phytosterols were prepared using reversed-phase HPLC in order to obtain the oxidized compounds of sitosterol, campesterol, stigmasterol and brassicasterol. 7-Hydroxy-, 7-keto-, 5,6-epoxy-, 4beta-hydroxy-, 4-ene-6-hydroxy-, 6-keto- and 5alpha,6beta-dihydroxyphytosterols were obtained as well as analogous compounds of cholesterol. The gas chromatographic properties as well as the electronic impact mass spectra of these compounds (as trimethylsilyl ether derivatives) were studied. These data were used to identify oxyphytosterols in a spread enriched in phytosterols: the oxyphytosterols represented no more than 68 microg/g of spread (about 0.08% of phytosterols were oxidised).     

Chemoenzymatic synthesis of GDP-azidodeoxymannoses: non-radioactive probes for mannosyltransferase activity

GDP-2-, 3-, 4- or 6-azidomannoses can be successfully prepared from the corresponding azidomannose-1-phosphates and GTP using the enzyme GDP-Mannosepyrophosphorylase (GDP-ManPP) from Salmonella enterica and may serve as useful probes for mannosyltransferase activity.