Biosynthesis of keratan sulfate: purification and properties of a galactosyltransferase from bovine cornea.

A soluble galactosyltransferase was purified 22,000-fold from bovine cornea. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-acetyl-d-glucosamine, α- and β-glucosaminides, bovine cornea and nasal septum agalactokeratan, and to glycoproteins containing terminal nonreducing N-acetylglucosaminyl units. When N-acetyl-d-glucosamine served as acceptor, the product formed by the cornea transferase contained galactose glycosidically linked to carbon atom 4 of N-acetyl-d-glucosamine; the same glycosidic linkage was found in [¹⁴C]keratan preparations isolated from reaction mixtures where keratan containing terminal nonreducing N-acetylglucosaminyl units served as acceptor. The cornea enzyme exhibited a markedly lower K_m with keratan than with N-acetyl-d-glucosamine. The physical and kinetic properties of the cornea galactosyltransferase and of the milk A-protein (A-protein + α-lactalbumin = lactose synthase), including modulations of acceptor specificity by α-lactalbumin, were compared. The results of these studies strongly suggest that the two glycosyltransferases are similar, if not identical. Efforts to demonstrate the presence of other soluble galactosyltransferases in cornea were unsuccessful; no change in the ratios of products formed with several acceptors was observed at any stage of purification. It is suggested that in bovine tissues a single galactosyltransferase participates in the synthesis of both high and low molecular weight galactosides including the assembly of the repeating disaccharide [O-β-galactopyranosyl-(1 → 4)-N-acetylglucosamine] of cornea keratan sulfate.     

Inhibition of microbial β-N-acetylhexosaminidases by 4-deoxy- and galacto-analogues of NAG-thiazoline

NAG-thiazoline is a well-established competitive inhibitor of two physiologically relevant glycosidase families—β-N-acetylhexosaminidases (GH20) and β-N-acetylglucosaminidases (GH84). Based on the different substrate flexibilities of these enzyme groups, we designed and synthesized the 4-deoxy derivative of NAG-thiazoline aiming at the selective inhibition of GH20 β-N-acetylhexosaminidases. One GH84 and two GH20 microbial glycosidases were employed as model enzymes for the inhibition assays. Surprisingly, the new compound 4-deoxy-thiazoline exhibited no activity inhibition with either of the enzyme families of interest. Unlike with the substrates, the 4-hydroxyl group of the inhibitor’s sugar ring seems to be crucial for binding the inhibitor to the active sites of these enzymes.     

β-N-Acetylhexosaminidase: What's in a name…?

β-N-Acetylhexosaminidases (EC 3.2.1.52, belonging to CAZy GH families 3, 20 and 84) have recently gained a lot of attention, not only due to their implication in human physiology and disease, but also due to their great potential in the enzymatic synthesis of carbohydrates and glycomimetics. GH family 20 β-N-acetylhexosaminidases, and GH family 3 and 84 β-N-acetylglucosaminidases from all kinds of organisms have been intensively studied from the point of view of their physiological roles, reaction mechanisms, structure and inhibition. Thanks to their outstanding substrate promiscuity, extracellular β-N-acetylhexosaminidases from filamentous fungi are able to cleave and transfer substrates bearing various functionalities, ranging from carboxylates, sulfates, acylations to azides, and even 4-deoxy glycosides. Thus, they have proved to be versatile biosynthetic tools for the preparation of both natural and modified hexosaminides under mild conditions with good yields.     

Alpha-secondary tritium isotope effects in the aqueous hydrolysis of glycopyranosides of N-acetyl-beta-D-glucosamine

Secondary tritium isotope effects were used to study the aqueous hydrolysis of a series of α- and β-glycopyranosides of N-acetyl-d-glucosamine. The magnitude of the secondary tritium isotope effects, and their dependence on the structure of the aglycone, are compatible with the carbonium ion mechanism suggested for the specific acid catalysis of these compounds by Piszkiewicz and Bruice (1–3). The secondary tritium isotope effect determined for the spontaneous hydrolysis of the p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside is not consistent with an intramolecular, nucleophilic displacement mechanism. A mechanism involving the equilibrium formation of a carbonium ion-anion pair is proposed. The relevance of these model studies to hydrolysis of oligosaccharides of N-acetyl-d-glucosamine by lysozyme is discussed.     

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-β-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two l-glutamine residues. A substance P derivative with an N-acetyl-d-glucosamine residue attached to the fifth or sixth l-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-d-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the l-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the l-glutamine residue of the peptide was not liberated by peptide-N⁴-(N-acetyl-β-d-glucosaminyl) asparagine amidase F.     

Effect of glucose and glucitol analogues on respiration by mycelia ofPuccinia graminis

Previous work has shown that the glucose analogue 2-deoxy-d-glucose (2dG) is extensively metabolized to a series of derivatives including 2-deoxy-d-glucitol (2dGol) by mycelia ofPuccinia graminis. In the present investigation, the time course of action of 2dG, 2dGol, and eight other glucose analogues as respiratory inhibitors ofP. graminis was determined by measuring the effect of each analogue on endogenous rates of oxygen uptake, using a manometric method. At a test concentration of 10 mM, the most potent inhibition was observed with 2dG, thio-β-d-glucose, and 2-fluoro-2-deoxy-d-glucose, each achieving ∼65% inhibition after 5 h; little or no inhibition was observed with 1-O-methyl-β-d-glucose, phenyl-β-d-glucose, 6-deoxyglucose, 3-O-methyl-d-glucose,N-acetyl-d-glucosamine, orl-glucose. However, mild inhibition (∼30% after 5 h) was observed with thed-glucitol (sorbitol) analogue 2dGol. It was concluded that the three inhibitory glucose analogues probably targeted central pathways of glucose metabolism, whereas 2dGol selectively inhibited an optional pathway associated with turnover of an endogenously generated pool ofd-glucose.     

The chain length of the glycans in bacterial cell walls (Short Communication)

The glycan chain length of peptidoglycan was measured by reduction with NaB³H₄ and isolation of the resulting muramitol, indicative of the length of the chains as biosynthesized, and glucosaminol, which measured the length of the chains after rupture by endo-β-N-acetylglucosaminidases. Measurement of the non-reducing terminal N-acetylglucosamine by Smith degradation confirmed the result.     

A remodeling system for the oligosaccharide chains on glycoproteins with microbial endo-β-N-acetylglucosaminidases

Endo-M, endo-β-N-acetylglucosaminidase from Mucor hiemalis, transferred the complex type oligosaccharide of sialoglycopeptide to partially deglycosylated proteins (N-acetylglucosamine-attached proteins), which were prepared by excluding high-mannose type oligosaccharides from glycoproteins with Endo-H, endo-β-N-acetylglucosaminidase from Streptomyces plicatus. This finding indicated that the high-mannose type oligosaccharides on glycoproteins can be changed to complex type ones by the transglycosylation activity of Endo-M. This is the first report of the establishment of a remodeling system for the different types of oligosaccharides on glycoproteins with microbial endo-β-N-acetylglucosaminidases having different substrate specificities. Endo-M is a powerful tool for the in vitro synthesis of glycoproteins containing complex type oligosaccharides from glycoproteins produced by yeast.     

The characterization of N-acetylglucosaminidases from the limpet Patella vulgata (L.)

The α- and β-N-acetylglucosaminidase activity of the limpet Patella vulgata (L.) is due to two enzymes. One of these enzymes hydrolyses both α- and β-N-acetylglucosaminidases and is referred to α,β-N-acetylglucosaminidase. The other is a β-N-acetylglucosaminidase (EC 3.2.1.30). Both enzymes have been isolated and characterized as glycoproteins containing 12% hexose, mainly galactose. The amino acid, neutral sugar and amino sugar content of the two enzymes is very similar, and the main difference lies in the presence of 9% sialic acid in β-N-acetylglucosaminidase. The molecular weight of α,β-N-acetylglucosaminidase is 217 000 and that of β-N-acetylglucosaminidase is 136 000. Evidence has been obtained for the presence of an additional sub-unit in the α,β-enzyme.     

The identification by x-ray crystallography of the site of attachment of an affinity label to hen egg-white lysozyme

Tetragonal crystals of hen egg-white lysozyme were treated with the active sitedirected irreversible inhibitor 2′,3′epoxypropyl β-glycoside of N-acetyl-d-glucosamine, β(1→4)-linked dimer. The crystals were examined by X-ray crystallography, and the results compared to those obtained from crystals of the reversible complex formed between hen egg-white lysozyme and the β-phenyl glycoside of GlcNAc β(1→4)GlcNAc. It is concluded that the GlcNAc β(1→4)GlcNAc moiety of the irreversible inhibitor occupies subsites B and C in the active site of the enzyme, and that the inhibitor is linked covalently to the enzyme through the carboxyl side-chain of Asp 52.     

Synthesis and antimicrobial activity of β-carboline derivatives with N2-alkyl modifications

β-Carbolines constitute a vast group of indole alkaloids and exhibit various biological actions. The objective of this study was to investigate the structure–activity relationships of β-carboline derivatives on in vitro inhibitory effects against clinically relevant microorganisms. A series of β-carboline dimers and their N²-alkylated analogues were therefore prepared and evaluated for their antimicrobial effects. Among these, a dimeric 6-chlorocarboline N²-benzylated salt exerted potent activity against Staphylococcus aureus at MICs of 0.01–0.05?μmol/mL. Our work highlights that N¹-N¹ dimerization and N²-benzylation significantly enhanced the antimicrobial effects of compounds.     

Structural and Kinetic Analysis of Bacillus subtilis N-Acetylglucosaminidase Reveals a Unique Asp-His Dyad Mechanism

Three-dimensional structures of NagZ of Bacillus subtilis, the first structures of a two-domain β-N-acetylglucosaminidase of family 3 of glycosidases, were determined with and without the transition state mimicking inhibitor PUGNAc bound to the active site, at 1.84- and 1.40-Å resolution, respectively. The structures together with kinetic analyses of mutants revealed an Asp-His dyad involved in catalysis: His²³⁴ of BsNagZ acts as general acid/base catalyst and is hydrogen bonded by Asp²³² for proper function. Replacement of both His²³⁴ and Asp²³² with glycine reduced the rate of hydrolysis of the fluorogenic substrate 4′-methylumbelliferyl N-acetyl-β-d-glucosaminide 1900- and 4500-fold, respectively, and rendered activity pH-independent in the alkaline range consistent with a role of these residues in acid/base catalysis. N-Acetylglucosaminyl enzyme intermediate accumulated in the H234G mutant and β-azide product was formed in the presence of sodium azide in both mutants. The Asp-His dyad is conserved within β-N-acetylglucosaminidases but otherwise absent in β-glycosidases of family 3, which instead carry a “classical” glutamate acid/base catalyst. The acid/base glutamate of Hordeum vulgare exoglucanase (Exo1) superimposes with His²³⁴ of the dyad of BsNagZ and, in contrast to the latter, protrudes from a second domain of the enzyme into the active site. This is the first report of an Asp-His catalytic dyad involved in hydrolysis of glycosides resembling in function the Asp-His-Ser triad of serine proteases. Our findings will facilitate the development of mechanism-based inhibitors that selectively target family 3 β-N-acetylglucosaminidases, which are involved in bacterial cell wall turnover, spore germination, and induction of β-lactamase.     

o-phthalaldehyde for the fluorometric assay of nonprotein amino compounds.

Procedures for the preparation of UDP-N-[1-¹⁴C]acetyl-d-glucosamine and UDP-N-[1-¹⁴C]acetyl-d-galactosamine with very high specific activities are deseribed. The overall yield based on the amount of [1-¹⁴C]acetate used is greater than 80%. The N-acetyl-d-glucosamine-α-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-d-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-d-glucosamine-α-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. d-glucosamine-α-1-phosphate is N-acetylated with [¹⁴C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-d-glucosamine pyrophosphorylase. UDP-N-[1-¹⁴C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-¹⁴C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Endo-beta-N-acetylglucosaminidases acting on carbohydrate moieties of glycoproteins: purification and properties of the two enzymes with different specificities from Clostridium perfringens.

Two endo-β-N-acetylglucosaminidases (C_I and C_I) acting on carbohydrate moieties of glycoproteins were highly purified from the culture fluid of Clostridium perfringens. C_I had the substrate specificity indistinguishable from that of endo-β-N-acetylglucosaminidase D from Diplococcus pneumoniae. C_II showed the specificity similar to that of endo-β-N-acetylglucosaminidase H from Streptomyces griseus but is distinct from the streptomyces enzyme with respect to the relative activity toward ovalbumin glycopeptides and Unit A glycopeptides of thyroglobulin. Both enzymes from C. perfringens were most active at neutral pH and were inhibited by p-chloromercuriphenylsulfonate.     

Molecular Cloning of the Gene Encoding an Outer-Membrane-Associated β-N-Acetylglucosaminidase Involved in Chitin Degradation System of Alteromonas sp. Strain O-7

The gene encoding β-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97 kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial β-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.     

Fractionation and molecular-weight determination of large polypeptides by gel filtration in guanidiniun chloride

The interaction of several N-acetyl-d-glucosamine analogs and of sialyl lactose with the lectin wheat germ agglutinin was studied by nuclear magnetic resonance. N-²H₃-acetyl-d-gluocosamine was synthesized and found to displace the N-acetyl methyl signal toward its free chemical shift in N-acetylglucosamine and N-acetylneuraminic acid demonstrating common binding sites for the latter two compounds. The N-acetyl methyl signal of the α-methylglucoside of N-acetylglucosamine could be titrated but a 3-deoxy analog could not, the latter exhibiting very weak binding and demonstrating the importance of the 3-OH group in the binding process. Sialyl lactose (an N-acetylneuraminic acid analog) was rather tightly bound to the lectin. N-F₃-acetyl-d-glucosamine was synthesized and its binding to the lectin was studied at pH 4, 4.5, 5.1 by ¹⁹F NMR. The two anomers were found to bind with nearly equal K_d′s but exhibited a pH and anomer dependent Δ (total bound chemical shift). The -CF₃ analog was found to bind considerably stronger to the lectin than the -CH₃ compound. The clear resolution of the α and β anomers of this molecule make it a very useful probe of the lectin binding site.     

Pochonicine,a polyhydroxylated pyrrolizidine alkaloid from fungus Pochonia suchlasporia var. suchlasporia TAMA 87 as a potent β-N-acetylglucosaminidase inhibitor

A new polyhydroxylated pyrrolizidine alkaloid designated as pochonicine (1) was isolated from a solid fermentation culture of the fungal strain Pochonia suchlasporia var. suchlasporia TAMA 87. The structure of 1 was determined using NMR and MS techniques as (1R*, 3S*, 5S*, 6S*, 7R*, 7a S*)-5-acetamidomethyl-3-hydroxymethyl-1,6,7-trihydroxypyrrolizidine. Pochonicine (1) showed potent inhibition against β-N-acetylglucosaminidases (GlcNAcases) of various organisms including insects, fungi, mammals, and a plant but no inhibition against β-glucosidase of almond, α-glucosidase of yeast, or chitinase of Bacillus sp. The GlcNAcase inhibitory activity of pochonicine (1) was comparable to nagstatin, a potent GlcNAcase inhibitor of natural origin.     

Synthesis and anti-CVB3 activity of 4-amino acid derivative substituted pyrimidine nucleoside analogues

Seven novel 4-amino acid derivative substituted pyrimidine nucleoside analogues were designed, synthesized, and tested for their anti-CVB3 activity. Initial biological studies indicated that among these 4-amino acid derivative substituted pyrimidine nucleoside analogues, 4-N-(2′-amino-glutaric acid-1′-methylester)-1-(2′- deoxy-2′-β-fluoro-4′-azido)-furanosyl-cytosine 2 exhibited the most potent anti-CVB activity (IC₅₀ = 9.3 μM). The cytotoxicity of these compounds has also been assessed. The toxicity of compound 2 was similar to that of ribavirin.     

Engineered N-acetylhexosamine-active enzymes in glycoscience

BackgroundIn recent years, enzymes modifying N-acetylhexosamine substrates have emerged in numerous theoretical studies as well as practical applications from biology, biomedicine, and biotechnology. Advanced enzyme engineering techniques converted them into potent synthetic instruments affording a variety of valuable glycosides.Scope of reviewThis review presents the diversity of engineered enzymes active with N-acetylhexosamine carbohydrates: from popular glycoside hydrolases and glycosyltransferases to less known oxidases, epimerases, kinases, sulfotransferases, and acetylases. Though hydrolases in natura, engineered chitinases, β-N-acetylhexosaminidases, and endo-β-N-acetylglucosaminidases were successfully employed in the synthesis of defined natural and derivatized chitooligomers and in the remodeling of N-glycosylation patterns of therapeutic antibodies. The genes of various N-acetylhexosaminyltransferases were cloned into metabolically engineered microorganisms for producing human milk oligosaccharides, Lewis X structures, and human-like glycoproteins. Moreover, mutant N-acetylhexosamine-active glycosyltransferases were applied, e.g., in the construction of glycomimetics and complex glycostructures, industrial production of low-lactose milk, and metabolic labeling of glycans. In the synthesis of biotechnologically important compounds, several innovative glycoengineered systems are presented for an efficient bioproduction of GlcNAc, UDP-GlcNAc, N-acetylneuraminic acid, and of defined glycosaminoglycans.Major conclusionsThe above examples demonstrate that engineering of N-acetylhexosamine-active enzymes was able to solve complex issues such as synthesis of tailored human-like glycoproteins or industrial-scale production of desired oligosaccharides. Due to the specific catalytic mechanism, mutagenesis of these catalysts was often realized through rational solutions.General significanceSpecific N-acetylhexosamine glycosylation is crucial in biological, biomedical and biotechnological applications and a good understanding of its details opens new possibilities in this fast developing area of glycoscience.     

The X-ray Crystal Structure of an Arthrobacter protophormiae Endo-β-N-Acetylglucosaminidase Reveals a (β/α)8 Catalytic Domain, Two Ancillary Domains and Active Site Residues Key for Transglycosylation Activity

Glycoside hydrolase family GH85 is a family of endo-β-N-acetylglucosaminidases that is responsible for the hydrolysis of β-1,4 linkage in the N,N-diacetylchitobiose core of N-linked glycans. The endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) is of particular interest, given its increasing use for the chemoenzymatic synthesis of bespoke N-glycans using N-glycan oxazolines as glycosyl donors. The E173Q variant of Endo-A is especially attractive for synthesis, as it is hydrolytically impaired but still able to catalyze N-glycan synthesis by transglycosylation using activated oxazoline donors. Here we present the three-dimensional structure of the A. protophormiae Endo-A E173Q variant, solved by multiple-wavelength anomalous scattering methods and refined at 1.8 Å resolution. The structure reveals that GH85 enzymes display a trimodular architecture in which a (β/α)₈ catalytic domain occurs with two ancillary β-sheet modules. The active centre is fully consistent with the known neighboring-group catalytic mechanism in which E173 acts as the catalytic acid/base for reaction via an oxazoline intermediate. Of note is the presence of an asparagine in the active centre, in a position likely to interact with the acetyl NH group that, in all other known families of glycosidase using this mechanism, is an aspartate or glutamate residue. The substrate-binding surface reveals an open topography, consistent with the ability to accept a large range of glycoprotein substrates and the ability to transglycosylate other acceptors. The three-dimensional structure of this important biocatalyst reveals that residues implicated in the enhancement of transglycosylation and synthetic capacity are proximal to the active centre, where they may act to favor binding of acceptor substrates.