Exploring specificity of glycosyltransferases: synthesis of new sugar nucleotide related molecules as putative donor substrates

We investigated the specificity of glycosyltransferases toward donor substrates in two complementary directions. First we prepared simple N-acetyl-alpha-D-glucosamine 1-diphosphates: methyl-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-diphosphate, benzyl-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-diphosphate, 4-phenylbutyl-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-diphosphate, by the coupling of the corresponding activated alkyl phosphates with N-acetyl-alpha-D-glucosamine 1-phosphate. These diphosphates as well as 2-acetamido-2-deoxy-alpha-D-glucopyranose 1-diphosphate, tested as donors of N-acetylglucosamine in a reaction catalyzed by Neisseria meningitidis N-acetylglucosaminyltransferase (LgtA), proved to be devoid of activity. Evaluated as inhibitors, only 2-acetamido-2-deoxy-alpha-D-glucopyranose 1-diphosphate showed some inhibitory activity with an IC50 value of 7 mM. In the second approach, we prepared sugar nucleotide mimics having the diphosphate bridge replaced by the oxycarbonylaminosulfonyl linker. The surrogate of GDP-Fuc was synthesized as a 9:1 alpha/beta anomeric mixture, in 40% yield, starting from chlorosulfonyl isocyanate, perbenzylated l-fucopyranose, and a guanosine derivative, protected on the exocyclic amine and secondary hydroxyl functions of ribose. Then two deprotection steps, hydrogenolysis and enzymatic hydrolysis catalyzed by penicillin G amidase afforded the target molecule to be tested as fucose donor with recombinant human alpha-(1-->3/4)-fucosyltransferase (FucT-III). Tested as a 4:1 alpha/beta anomeric mixture, both in the absence and in the presence of cationic cofactors, this new guanosine fucose conjugate proved to be ineffective. Its inhibitory activity toward FucT-III evaluated through a competition fluorescence assay was very poor (IC50 value of 20 mM). The surrogate of UDP-GlcNAc that was already known as its protected acetylated derivative, tested as N-acetylglucosamine donor with LgtA in the presence of Mn(2+) turned out not to be active either.     

Concentration measurements of sucrose and sugar surfactants solutions by using the 1H NMR ERETIC method

The ERETIC method has been used to determine precise concentrations of aqueous solutions of sucrose and sugar surfactants, namely octyl glucoside and fatty acid sucrose esters by (1)H NMR spectroscopy. The effects of NMR tuning, acquisition parameters, and spectrum processing on the measurement have been assessed in these particular cases. The linearity upholds over the whole concentration range, with both sucrose and octyl glucoside, whatever the physicochemical phenomena occurring, either an increasing viscosity or the micellization of the surfactant. For sucrose solutions, an accuracy of 2% is measured for concentrations between 0.1 and 200 mmol/L, which is consistent with literature data.     

A new sensitive colorimetric assay for peroxidase using 3,3'-diaminobenzidine as hydrogen donor

A new simple colorimetric assay for measurement of peroxidase activity using 3,3′-diaminobenzidine tetrahydrochloride as hydrogen donor is described. The DAB is stable under the usual assay conditions, and its rate of auto-oxidation is negligible. Under optimal conditions, a linear relationship is found between peroxidase concentration and the rate of oxidation of 3,3′-diaminobenzidine tetrahydrochloride (ΔA_405nm/min). Using horseradish peroxidase, the DAB method appears more sensitive than the o-dianisidine and the guaiacol assays for peroxidase. This method can also be used for measurement of peroxidase activity in tissue fractions.     

A convenient synthesis of glycosyl chlorides from sugar hemiacetals using triphosgene as the chlorine source

Treating partially protected sugar hemiacetals with triphosgene in THF results in the formation of glycosyl chlorides. The method is compatible with acid-sensitive isopropylidene protecting groups in the hemiacetal substrates.     

Glycoprotein synthesis in lysolecithin-treated cells using sugar nucleotides as glycosyl donors

Summary The 3T3 cells were treated with 50 μg/ml lysolecithin (LL) followed by the addition of exogenously supplied radiolabelled sugar nucleotides to serve as direct glycosyl donors. These were found to be 1.5 to 3.0 times more active than untreated cells in their glycosyl transferase activities depending on the particular sugar nucleotide used. Mannosyl transferase activity was not inhibited by 2-deoxyglucose or mannose-1-phosphate, indicating that the sugar nucleotide remained intact throughouth the assay period. Preincubation of the cells with tunicamycin caused an 85% decrease in mannosyl transfer, which suggested that the normal pathway of glycosylation via lipid intermediates was still operable in the treated cells. Fractionation of control and LL-treated cells after incubation with UDP[³H]galactose revealed that only microsomal and cytosolic proteins from the treated cells were radioactive. Thus, intracellular labelling of permeabilized cells was allowed. About 80% of the radiolabeled product was glycoprotein in nature, based upon its solubilization with pronase. This work was supported by institutional funds granted by Texas Woman's University.     

Glycoprotein synthesis in lysolecithin-treated cells using sugar nucleotides as glycosyl donors

The 3T3 cells were treated with 50 mu g/ml lysolecithin (LL) followed by the addition of exogenously supplied radiolabelled sugar nucleotides to serve as direct glycosyl donors. These were found to be 1.5 to 3.0 times more active than untreated cells in their glycosyl transferase activities depending on the particular sugar nucleotide used. Mannosyl transferase activity was not inhibited by 2-deoxyglucose or mannose-1-phosphate, indicating that the sugar nucleotide remained intact throughout the assay period. Preincubation of the cells with tunicamycin caused an 85% decrease in mannosyl transfer, which suggested that the normal pathway of glycosylation via lipid intermediates was still operable in the treated cells. Fractionation of control and LL-treated cells after incubation with UDP[3H]galactose revealed that only microsomal and cytosolic proteins from the treated cells were radioactive. Thus, intracellular labelling of permeabilized cells was allowed. About 80% of the radiolabeled product was glycoprotein in nature, based upon its solubilization with pronase.     

A new method for the synthesis of N2-alkylguanosines using Mitsunobu reaction as a key step

Peracetylated guanosine was reacted with POCl3 to give an 2-acetamido-6-chloro-9H-purine derivative, which was condensed with primary or secondary alcohols to give N2-alkylated analogues. The products were treated with mercaptoethanol in the presence of sodium methoxide to afford N2-alkylguanosines.     

A new method for the synthesis of 2'-O-benzyladenosine using Mitsunobu reaction

A new method to introduce a benzyl group onto the 2'-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3'-O-benzoylriboside and its 5'-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2'-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent used. The product was converted to adenosine, indicating that the stereochemistry at C-2' is retained.     

Evidence for circadian regulation of starch and sucrose synthesis in sugar beet leaves

Starch accumulation and sucrose synthesis and export were measured in leaves of sugar beet (Beta vulgaris L.) during a period of prolonged irradiance in which illumination was extended beyond the usual 14-hour day period. During much of the 14-hour day period, approximately 50% of the newly fixed carbon was distributed to sucrose, about 40% to starch, and less than 10% to hexose. Beginning about 2 hours before the end of the usual light period, the portion of newly fixed carbon allocated to sucrose gradually increased, and correspondingly less carbon went to starch. By the time the transition ended, about 4 hours into the extension of the light period, nearly 90% of newly fixed carbon was incorporated into sucrose and little or none into starch. Most of the additional sucrose was exported. Gradual cessation of starch accumulation was not the result of a futile cycle of simultaneous starch synthesis and degradation. Neither was it the result of a decrease in the extractable activity of adenosine diphosphoglucose pyrophosphorylase or phosphoglucose isomerase, enzymes important in starch synthesis. Nor was there a notable change in control metabolites considered to be important in regulating starch synthesis. Starch accumulation appeared to decrease markedly because of an endogenous circadian shift in carbon allocation, which occurred in preparation for the usual night period and which diverted carbon from the chloroplast to the cytosol and sucrose synthesis.     

A new colorimetric method for determining the isomerization activity of sucrose isomerase

A new colorimetric method for determining the isomerization activity of sucrose isomerase was developed. This colorimetric method is based on the enzymatic reactions of invertase and glucose oxidase-peroxidase (GOD-POD). The main scheme for assaying sucrose isomerase activity is to degrade sucrose in the reaction mixture to glucose and fructose by invertase and to detect the concentration of glucose generated using GOD-POD. The concentrations of trehalulose and isomaltulose, reaction products of sucrose isomerase, are calculated from the concentration of glucose. This method allows rapid and accurate determination of the isomerization activity of sucrose isomerase without inhibition by hydrolysis activity.     

A new method to assess air pollution using lichens as bioindicators

Lichens are increasingly used worldwide as air quality biomonitors because they are efficient, easy and cheap, but validation studies of the methodology are scarce. Three foliose lichen biomonitoring methods were compared by field tests (in the tropical urban habitat of San José, Costa Rica) and laboratory simulations: (1) the 100 uniform squares template traditionally used in North America, (2) the European 200 uniform points template and (3) a new computer-generated random points template (10 x 20 cm) in two versions: 100 points and 50 points. Repeated measurement by the same observer causes a variation of 2-14% and the templates' error is 0.2-11%. We recommend the 100 random point template (applied to four sides of trunk) for ecological studies and the 50 random points template (applied to side with greatest lichen cover) for biomonitoring because it reduces time and costs by nearly 50% but still has acceptable reliability values.     

Enzymatic synthesis of sucrose octaacetate using a novel alkaline protease

Acylation of 0.5 g sucrose with 1.2 ml acetic anhydride was carried out in 2 ml two-solvent medium of anhydrous pyridine/n-hexane (1:1, v/v) using 0.2 g crude protease from Serratia sp. Sucrose octaacetate was the sole product and more than 90% sucrose was converted in 24 h at 30°C. The purity of sucrose octaacetate reached 100%, via a simple purification method of alcohol/water washing and centrifugation.     

A new mechanism and kinetic model for the enzymatic synthesis of short-chain fructooligosaccharides from sucrose

A kinetic model based on a ping-pong mechanism was developed under the steady-state hypothesis to account for the short-chain fructooligosaccharides (sc-FOS) synthesis using the commercial cellulolytic enzyme preparation, Rohapect CM. This new mechanism takes into account the interactions between the enzyme species and potential substrates (sucrose and sc-FOS) as a single complex reaction, allowing a better understanding of the reaction kinetics.The initial reaction rate laws appropriately describe the kinetic profiles of the examined substrates. Whereas sucrose exhibited Michaelis–Menten behavior with substrate inhibition, 1-kestose and nystose followed Michaelis–Menten and sigmoid enzyme kinetics. In addition, the enzyme was competitively inhibited by glucose and exhibited significant hydrolytic activity in the presence of nystose.The overall model was simultaneously fitted to experimental data from three initial sucrose concentrations (0.5, 1.5 and 2.1 M) using a multi-response regression with kinetic parameters that have biochemical relevance and are independent of the enzyme concentration. According to the model, sucrose acts almost exclusively as a fructosyl donor substrate. The mathematical development described herein is expected to be suitable for modeling similar enzymatic reaction systems.     

Fructansucrase enzymes and sucrose analogues: A new approach for the synthesis of unique fructo-oligosaccharides

Fructansucrase enzymes of lactic acid bacteria use the cheap compound sucrose (Glc–Fru) to synthesize a variety of poly- and oligosaccharide products. Recently, it has been shown that a variety of sucrose analogues (e.g. Gal–Fru, Man–Fru) can be efficiently synthesized. This has exciting potential for the development of novel (fructo) oligosaccharide derivatives.     

Fructansucrase enzymes and sucrose analogues: A new approach for the synthesis of unique fructo-oligosaccharides

Fructansucrase enzymes of lactic acid bacteria use the cheap compound sucrose (Glc-Fru) to synthesize a variety of poly- and oligosaccharide products. Recently, it has been shown that a variety of sucrose analogues (e.g. Gal-Fru, Man-Fru) can be efficiently synthesized. This has exciting potential for the development of novel (fructo) oligosaccharide derivatives.     

Evolution of sucrose synthesis

Cyanobacteria and proteobacteria (purple bacteria) are the only prokaryotes known to synthesize sucrose (Suc). Suc-P synthase, Suc-phosphatase (SPP), and Suc synthase activities have previously been detected in several cyanobacteria, and genes coding for Suc-P synthase (sps) and Suc synthase (sus) have been cloned from Synechocystis sp. PCC 6803 and Anabaena (Nostoc) spp., respectively. An open reading frame in the Synechocystis genome encodes a predicted 27-kD polypeptide that shows homology to the maize (Zea mays) SPP. Heterologous expression of this putative spp gene in Escherichia coli, reported here, confirmed that this open reading frame encodes a functional SPP enzyme. The Synechocystis SPP is highly specific for Suc-6(F)-P (K(m) = 7.5 microM) and is Mg(2+) dependent (K(a) = 70 microM), with a specific activity of 46 micromol min(-1) mg(-1) protein. Like the maize SPP, the Synechocystis SPP belongs to the haloacid dehalogenase superfamily of phosphatases/hydrolases. Searches of sequenced microbial genomes revealed homologs of the Synechocystis sps gene in several other cyanobacteria (Nostoc punctiforme, Prochlorococcus marinus strains MED4 and MIT9313, and Synechococcus sp. WH8012), and in three proteobacteria (Acidithiobacillus ferrooxidans, Magnetococcus sp. MC1, and Nitrosomonas europaea). Homologs of the Synechocystis spp gene were found in Magnetococcus sp. MC1 and N. punctiforme, and of the Anabaena sus gene in N. punctiforme and N. europaea. From analysis of these sequences, it is suggested that Suc synthesis originated in the proteobacteria or a common ancestor of the proteobacteria and cyanobacteria.