Retention of anomeric form in lysozyme-catalyzed reaction

A lysozyme-catalyzed reaction is initiated by a cleavage of the beta-1, 4-glucosaminide linkage, followed by hydration and transglycosylation. Since all glycosides produced by transglycosylation have beta-glycosidic linkages between the sugar and the acceptor moieties, the lysozyme-catalyzed reaction has been classified as an anomer-retention reaction. However, there is no experimental evidence on the anomer retention of the new reducing residue produced by the hydrolysis of the substrate. In the present study, an attempt was made to determine the anomeric form of the GlcNAc residue at the reducing end in nascent hydrolytic products. The anomeric forms of the enzymatic products were separated and quantitatively analyzed by high-performance liquid chromatography. The amounts of alpha- and beta-anomers in the product were plotted against the reaction time. Computer analysis of the experimental data indicated that the nascent hydrolytic product takes only the beta-anomeric form and that the alpha-anomer is formed from beta-anomer by mutarotation.     

Sugar-linked biodegradable polymers: Regio-specific ester bonds of glucose hydroxyls in their reaction with maleic anhydride functionalized polystyrene and elucidation of the polymer structures formed

In the development of sugar-linked synthetic polymers as biodegradable polymers, it is imperative to know the variety of polymer structures formed by the reaction of a multi-functional sugar molecule with the functionalized synthetic polymer on which the sugar is to be anchored. Enzymes produced by the microorganisms causing the polymer to biodegrade can be sensitive to the particular type of sugar hydroxyl utilized (such as anomeric, primary, or secondary hydroxyl group) for getting anchored to the polymer. In this paper, we present synthesis of regio-specific ester derivatives of glucose with anhydride, functionalized polymers, i.e., ester formation specifically with the anomeric, primary or secondary hydroxyls of glucose. Characterization of these different esters groups was done using FTIR spectroscopy; each ester peak was further deconvoluted to yield its different components. For this purpose, we studied the reactions of d-glucose, 6-O-trityl glucose, methyl glucoside, 1,2-5,6-diisopropylidene-d-glucose, and 1,2,3,4-tetraacetyl-d-glucose with maleic anhydride functionalized polystyrene (PSMAH). In this study, the primary hydroxyl of glucose was found to be even more reactive than the anomeric hydroxyl. The peaks at 1716, 1725, and 1729–1737 cm⁻¹ were assigned to the ester carbonyl of the anomeric, primary, and secondary hydroxyls of glucose (C2, C3, and C4), respectively. An attempt was made to quantify the extent to which the different polymer structures are formed in a particular reaction by taking ratios of non-variable reference peaks (polystyrene peak at 1493 cm⁻¹) and variable peaks caused by the reaction (the residual anhydride carbonyl at 1780 cm⁻¹).     

Chromium in biological systems, I. Some observations on glucose tolerance factor in yeast

Glucose tolerance factor (GTF) has been isolated from a commercially available yeast extract powder, by a simple procedure under mild conditions. This cationic yellow material enhances considerably CO2 production in several yeast strains, after a lag time which can be eliminated by preincubation with glucose. The enhancement of CO2 production by GTF is not specific for glucose, and its effect on galactose raises the possibility that it influences the transport of the sugar to the cells. The ineffectiveness of GTF on cell free extract and the results of a Michaelis plot for CO2 production support this hypothesis.     

Substrate-induced conformational changes in glycosyltransferases

Oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans are synthesized by glycosyltransferases by the transfer of specific glycosyl moieties from activated sugar-nucleotide donors to specific acceptors. Structural studies on several of these enzymes have shown that one or two flexible loops at the substrate-binding site of the enzymes undergo a marked conformational change from an open to a closed conformation on binding the donor substrate. This conformational change, in which the loop acts as a lid covering the bound donor substrate, creates an acceptor-binding site. After the glycosyl unit is transferred from the donor to the acceptor, the saccharide product is ejected and the loop reverts to its native conformation, thereby releasing the remaining nucleotide moiety. The specificity of the sugar donor is determined by a few residues in the sugar-nucleotide-binding pocket of the enzyme that are conserved among the family members from different species.     

Glycosylated analogs of formaecin I and drosocin exhibit differential pattern of antibacterial activity

The synthetic glycopeptides are interesting model systems to study the effect of O-glycosylation in modulating their function and structure. A series of glycosylated analogs of two antibacterial peptides, formaecin I and drosocin, were synthesized by varying the nature of sugar and its linkage with bioactive peptides to understand the influence of structure variation of glycosylation on their antibacterial activities. Higher antibacterial activities of all glycopeptides compared to their respective non-glycosylated counterparts emphasize in part the importance of sugar moieties in functional implications of these peptides. The consequences of the unique differences among the analogs were apparent on their antibacterial activities but not evident structurally by circular dichroism studies. We have shown that differently glycosylated peptides exhibit differential effect among each other when tested against several Gram-negative bacterial strains. The change of monosaccharide moiety and/or its anomeric configuration in formaecin I and drosocin resulted into decrease in the antibacterial activity in comparison to that of the native glycopeptide, but the extent of decrease in antibacterial activity of glycosylated drosocin analogs was less. Probably, the variation in peptide conformation arising due to topological dissimilarities among different sugars in the same peptide resulting in possible modulation in binding properties appears to be responsible for differences in their antibacterial activities. Indeed, these effects of glycosylation are found to be sequence-specific and depend in the milieu of amino acid residues. Interestingly, none of the carbohydrate variants affected the basic property of these peptides, which is non-hemolytic and non-toxicity to eukaryotic cells.     

Glycosylation of macrolide antibiotics. Purification and kinetic studies of a macrolide glycosyltransferase from Streptomyces antibioticus

The oleD gene has been identified in the oleandomycin producer Streptomyces antibioticus and it codes a macrolide glycosyltransferase that is able to transfer a glucose moiety from UDP-glucose (UDP-Glc) to many macrolides. The glycosyltransferase coded by the oleD gene has been purified 371-fold from a Streptomyces lividans clone expressing this protein. The reaction product was isolated, and its structure determined by NMR spectroscopy. The kinetic mechanism of the reaction was analyzed using the macrolide antibiotic lankamycin (LK) as substrate. The reaction operates via a compulsory order mechanism. This has been shown by steady-state kinetic studies and by isotopic exchange reactions at equilibrium. LK binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated lankamycin (GS-LK). A pH study of the reaction was performed to determine values for the molecular pK values, suggesting possible amino acid residues involved in the catalytic process.     

Improved anomeric selectivity for the aroylation of sugars

By manipulating the solvent and using bulky TMEDA as a base, good yields and improved anomeric selectivities were obtained for the aroylation of D-glucose over similar esterifications using pyridine. The reaction has been extended to mannose and the beta-anomer of pergalloylated mannose was predominantly obtained in one step by direct aroylation of the parent sugar.     

Enzymatic attachment of glycosaminoglycan chain to peptide using the sugar chain transfer reaction with endo-beta-xylosidase.

Endo-beta-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-beta-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-beta-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-beta-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-beta-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan.     

Regulation of self-glycosylation of reversibly glycosylated polypeptides from Solanum tuberosum

Reversibly glycosylated polypeptides (RGPs) belong to a family of self-glycosylating proteins believed to be involved in plant polysaccharide synthesis. The precise function of these enzymes remains to be elucidated. Our results showed that the RGP 38-kDa subunit is phosphorylated in potato extracts ( Solanum tuberosum L.). An increase in the self-glycosylation of Solanum tuberosum RGP (StRGP) 38-kDa subunit was observed after alkaline phosphatase (AP) treatment. Our results suggest that phosphorylation of StRGP appears to regulate its self-glycosylation. It was determined that when the StRGP reaction was carried out in the presence of UDP-[¹⁴C]Glc as the sugar donor and then 1 m M UDP was added in a chase-out experiment, radioactive UDP-Glc was obtained indicating that StRGP reaction seems to be reversible. The anomeric configuration of transferred sugars to StRGP protein was also studied.     

Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.     

Structural studies of the capsular polysaccharide of Klebsiella type 52

The structure of the capsular polysaccharide from Klebsiella Type 52 has been investigated. Methylation analysis, characterization by gas-liquid chromatography-mass spectrometry of oligosaccharide derivatives obtained on partial hydrolysis of the methylated polysaccharide with acid, and specific degradation of the methylated polysaccharide by successive treatments with base and acid followed by characterization of the product, were the principal methods used. The polysaccharide is composed of hexasaccharide repeating-units containing D-glucuronic acid, D-galactose, and L-rhamnose, in the ratios 1:3:2. A structure for these units, disregarding the anomeric natures of the sugar residues, is proposed.     

Synthesis of 1,2,3-triazolo-linked octyl (1→6)-α-D-oligomannosides and their evaluation in mycobacterial mannosyltransferase assay

The synthesis of conjugates consisting of two or three mannose units interconnected by a 1,2,3-triazole linker installed by the "click" reaction is reported. These conjugates were evaluated in mycobacterial mannosyltransferase (ManT) assay. Detailed analysis of the reaction products showed that these compounds with triazole linker between sugar moieties were tolerated by the enzyme, which elongated them by one or two sugar units with α-(1→6) linkage. The effectiveness of this transfer was reduced in comparison to that observed for the acceptor analogues containing a glycosidic linkage, but still, this is the first report on such unnatural compounds serving as substrates for mycobacterial ManT. The ability of the studied compounds to function as acceptors for the ManT suggests that the relative distance and spatial orientation of acceptor octyl hydrophobic aglycone (optimal length for the ManT) and free primary C-6 hydroxy group of the nonreducing terminal mannose unit (to which glycosyl residue is transferred by the mycobacterial ManT) are important for ManT activity, but at the same time, their variations are tolerated by the enzyme in a relatively wide range.     

Saturation–transfer–difference NMR to characterize substrate binding recognition and catalysis of two broadly specific glycoside hydrolases

Saturation–transfer–difference NMR spectroscopy (STD NMR) is used to delineate noncovalent enzyme–substrate interactions of β-glycosidases from Pyrococcus furiosus and Aspergillus fumigatus under binding-only conditions at low temperatures, and during catalysis. Glucopyranosyl and galactopyranosyl moieties display a distinct pattern of multiple contacts with each active site, revealing enzyme-specific elements of recognition and portraying the global binding effect caused by single-site modification of the substrate, at carbon 4. The glucopyranose leaving group of cellobiose or lactose shows small relative STD effects except for the anomeric carbon, particularly in the -form. Its replacement in β-glucosides by an alcohol leaving group strongly affects sugar binding in the proximal enzyme subsite. A combination of STD effects of substrate and product, produced by the catalytic event or added exogenously, characterizes subsite binding during cellobiose hydrolysis.     

Electrostatic guidance of glycosyl cation migration along the reaction coordinate of uracil DNA glycosylase

The DNA repair enzyme uracil DNA glycosylase has been crystallized with a cationic 1-aza-2'-deoxyribose-containing DNA that mimics the ultimate transition state of the reaction in which the water nucleophile attacks the anomeric center of the oxacarbenium ion-uracil anion reaction intermediate. Comparison with substrate and product structures, and the previous structure of the intermediate determined by kinetic isotope effects, reveals an exquisite example of geometric strain, least atomic motion, and electrophile migration in biological catalysis. This structure provides a rare opportunity to reconstruct the detailed structural transformations that occur along an enzymatic reaction coordinate.     

Anomeric specificity and protein-substrate interactions support the 3D model for the hemagglutinin-neuraminidase from sendai virus.

The 3D structure of paramyxovirus hemagglutinin-neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the hemagglutinin-neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase.     

Synthesis of Iodoaminoimidazole Arabinoside (IAIA): A Potential Reductive Metabolite of the Spect Imaging Agent,Iodoazomycin Arabinoside (IAZA)

Abstract

The stereospecific synthesis of 1-(5-deoxy-5-iodo-α-D-arabino-furanosyl)-2-aminoimidazole (iodoaminoimidazole arabinoside: IAIA, 2) is described. The reaction of the protected sugar bromide (8) and trifluoroacetamidoimidazole (10B) gave the coupled product (11B), which gave the free nucleoside (4) on deblocking. It was identical with AIA obtained by reduction of AZA (azomycin arabinoside), whose anomeric configuration was found to be a by the X-ray crystallography.     

Specificity of the glycolipid transfer protein from pig brain

Lipid specificity has been studied in the lipid transfer reaction facilitated by the glycolipid transfer protein from pig brain. The lipid transfer was measured by determining the transfer of a radioisotopically labeled lipid from donor liposomes to either acceptor liposomes or mitochondria. Whenever possible, the liposomes contained 1 mol % of the lipid whose transfer was under study. The transfer protein accelerates the transfer of glucosylceramide, galactosylceramide (GalCer), lactosylceramide (LacCer), galactosylceramide 3-sulfate, globotriaosylceramide, LacCer sulfate, sialosyl-LacCer, globotetraosylceramide, and globopentaosylceramide. An inverse relationship is found between the length of sugar chains in glycosphingolipids and the transfer rates. In addition to the glycosphingolipids, the transfer protein facilitates the transfer of galactosyldiacylglycerol, digalactosyldiacylglycerol, glucosyldiacylglycerol, and diglucosyldiacylglycerol. The protein does not facilitate the transfer of dimannosyldiacylglycerol. The transfer of periodate-oxidized and subsequently reduced derivatives of GalCer and LacCer is facilitated by the transfer protein. The derivatives of GalCer are transferred at lower rates than GalCer, whereas the derivatives of LacCer are transferred at higher rates than LacCer. The transfer protein does not facilitate the transfer of phosphatidylcholine, phosphatidylinositol, cholesterol, or cholesteryloleate. These results suggest that the glycolipid transfer protein from pig brain has specificity to hydroxyl groups present in the sugar residue directly linked to either ceramide or diacylglycerol. The presence of glucose or galactose linked to these hydrophobic moieties makes the glycolipid transferable by the protein.     

Structural dissection of the reaction mechanism of cellobiose phosphorylase

Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 A (1 A=0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established.     

A “biphasic glycosyltransferase high-throughput screen” identifies novel anthraquinone glycosides in the diversification of phenolic natural products

The sugar moieties of many glycosylated small molecule natural products are essential for their biological activity. Glycosyltransferases (GTs) are enzymes responsible for installing these sugar moieties on a variety of biomolecules. Many GTs active on natural products are inherently substrate promiscuous and thus serve as useful tools in manipulating natural product glycosylation to generate new combinations of sugar units (glycones) and scaffold molecules (aglycones) in a process called glycodiversification. It is important to have an effective screening tool to detect the activity of promiscuous enzymes and their resulting glycoside products. Toward this aim, we developed a strategy for screening natural product GTs in a high-throughput fashion enabled by rapid isolation and detection of chromophoric or fluorescent glycosylated natural products. This involves a solvent extraction step to isolate the resulting polar glycoside product from the unreacted aglycone acceptor substrate and the detection of the formed glycoside by the innate absorbance or fluorescence of the aglycone moiety. Using our approach, we screened a collection of natural product GTs against a panel of precursors to therapeutically important molecules. Three GTs showed previously unreported promiscuity toward anthraquinones resulting in novel ε-rhodomycinone glycosides. Considering the pharmaceutical value of clinically used anthraquinone glycosides that are biosynthesized from an ε-rhodomycinone precursor, and the significance that the sugar moiety has on the biological activity of these drugs, our results are of particular importance toward the glycodiversification of therapeutics in this class. The GTs identified and the novel compounds they produce show promise toward new biocatalytic tools and therapeutics.     

Installation of the pyrrolyl-2-carboxyl pharmacophore by CouN1 and CouN7 in the late biosynthetic steps of the aminocoumarin antibiotics clorobiocin and coumermycin A1

The 5-methyl-2-pyrrolylcarbonyl moiety of the aminocoumarin antibiotics clorobiocin and coumermycin A1 is the key pharmacophore for targeting the ATP-binding site of GyrB for inhibition of the bacterial type-II topoisomerase DNA gyrase. During the late stage of clorobiocin and coumermycin A1 biosynthesis, the pyrrolyl-2-carboxyl group is transferred from the peptidyl carrier proteins Clo/CouN1 to the 3'-hydroxyl of the 4-methoxy-L-noviosyl scaffold by the action of the acyltransferases Clo/CouN7. CouN1 and CouN7 have now been heterologously expressed and purified from Escherichia coli. The apo form of CouN1 is converted to the acyl-holo form by loading with pyrrolyl-2-carboxyl-S-pantetheinyl moieties from synthetic pyrrolyl- and 5-methylpyrrolyl-CoAs by the action of the phosphopantetheinyl transferase Sfp. CouN7 acts as an acyltransferase, moving the pyrrole acyl moieties from CouN1 to the noviose sugar of descarbamoylnovobiocin. When the 5-methylpyrrolyl-2-carboxyl-thioester of CouN1 is the cosubstrate, the in vitro product differs from clorobiocin only in a CH3 for Cl group change on the coumarin ring. Double transfer of this acyl moiety by CouN7 to the penultimate intermediate in coumermycin A1 assembly completes that antibiotic biosynthetic pathway.