Crystal Structure of Helicobacter pylori Pseudaminic Acid Biosynthesis N-Acetyltransferase PseH: Implications for Substrate Specificity and Catalysis

Helicobacter pylori infection is the common cause of gastroduodenal diseases linked to a higher risk of the development of gastric cancer. Persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. It belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. The crystal structure of the PseH complex with cofactor acetyl-CoA has been determined at 2.3 Å resolution. This is the first crystal structure of the GNAT superfamily member with specificity to UDP-4-amino-4,6-dideoxy-β-L-AltNAc. PseH is a homodimer in the crystal, each subunit of which has a central twisted β-sheet flanked by five α-helices and is structurally homologous to those of other GNAT superfamily enzymes. Interestingly, PseH is more similar to the GNAT enzymes that utilize amino acid sulfamoyl adenosine or protein as a substrate than a different GNAT-superfamily bacterial nucleotide-sugar N-acetyltransferase of the known structure, WecD. Analysis of the complex of PseH with acetyl-CoA revealed the location of the cofactor-binding site between the splayed strands β4 and β5. The structure of PseH, together with the conservation of the active-site general acid among GNAT superfamily transferases, are consistent with a common catalytic mechanism for this enzyme that involves direct acetyl transfer from AcCoA without an acetylated enzyme intermediate. Based on structural homology with microcin C7 acetyltransferase MccE and WecD, the Michaelis complex can be modeled. The model suggests that the nucleotide- and 4-amino-4,6-dideoxy-β-L-AltNAc-binding pockets form extensive interactions with the substrate and are thus the most significant determinants of substrate specificity. A hydrophobic pocket accommodating the 6’-methyl group of the altrose dictates preference to the methyl over the hydroxyl group and thus to contributes to substrate specificity of PseH.     

O acetylation of the enterobacterial common antigen polysaccharide is catalyzed by the product of the yiaH gene of Escherichia coli K-12

The carbohydrate component of the enterobacterial common antigen (ECA) of Escherichia coli K-12 occurs primarily as a water-soluble cyclic polysaccharide located in the periplasm (ECA(CYC)) and as a phosphoglyceride-linked linear polysaccharide located on the cell surface (ECA(PG)). The polysaccharides of both forms are comprised of the amino sugars N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc). These amino sugars are linked to one another to form trisaccharide repeat units with the structure -->3-alpha-D-Fuc4NAc-(1-->4)-beta-D-ManNAcA-(1-->4)-alpha-D-GlcNAc-(1-->. The hydroxyl group in the 6 position of the GlcNAc residues of both ECA(CYC) and ECA(PG) are nonstoichiometrically esterified with acetyl groups. Random transposon insertion mutagenesis of E. coli K-12 resulted in the generation of a mutant defective in the incorporation of O-acetyl groups into both ECA(CYC) and ECA(PG). This defect was found to be due to an insertion of the transposon into the yiaH locus, a putative gene of unknown function located at 80.26 min on the E. coli chromosomal map. Bioinformatic analyses of the predicted yiaH gene product indicate that it is an integral inner membrane protein that is a member of an acyltransferase family of enzymes found in a wide variety of organisms. The results of biochemical and genetic experiments presented here strongly support the conclusion that yiaH encodes the O-acetyltransferase responsible for the incorporation of O-acetyl groups into both ECA(CYC) and ECA(PG). Accordingly, we propose that this gene be designated wecH.     

Synthesis and glycan priming activity of acetylated disaccharides

Five disaccharides related in structure to the glycans of vertebrate mucins have been chemically synthesized using orthogonal blocking, coupling and deblocking techniques. These include 2-naphthylmethyl 3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-( 1 --> 4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-beta-D-glucopyranoside (6), 2-naphthylmethyl 2-aceta-mido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl-(1 --> 3)-2,4,6-tri-O-acetyl-beta-D-galactopyranoside (14), 2-naph-thylmethyl2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1 --> 3)-2-acetamido-4,6-di- O-acetyl-2-deoxy-alpha-D-galactopyranoside (20), 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl-(1 --> 3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside (23) and 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glu-copyranosyl-(1 --> 6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside (27). These per-O-acetylated compounds were fed to U-937 cells to test their ability to prime oligosaccharide synthesis, inhibit glycoprotein biosynthesis and alter adhesion to E-selectin expressed on endothelial cells. The results show that 6, 14, and 20 served as substrates for oligosaccharide synthesis. The generation of glycoside-primed glycans altered the formation of glycoproteins on the cell surface and inhibited cell adhesion dependent on E-selectin.     

Synthesis of a novel asparagine-linked heptasaccharide structure via p-methoxybenzyl-assisted beta-mannosylation

Synthesis of a core heptasaccharide asparagine N4-[alpha-D-mannopyranosyl-(1 --> 6)-[(alpha-D-mannopyranosyl)-(1 --> 3)]-[(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 --> 2)]-(beta-D-mannopyranosyl)-(1 --> 4)-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 --> 4)-[(alpha-L-fucopyranosyl)-(1 --> 6)]-2-acetamido-2-deoxy-beta-D-glucopyranosyl]-L-asparagine (1a) found from CHO glycosylation mutant cell LEC 14 is described. The structure of 1a is highly novel in terms of the presence of an extra GlcNAc residue linked to the 2-position of beta-linked mannose. The synthesis was performed using p-methoxybenzyl-assisted intramolecular aglycon delivery as the key transformation. 4,6-O-TIDPS-protected thiomannoside methyl 2-O-p-methoxybenzyl-4,6-O-(1,1,3,3-tetraisopropyl)disiloxanylid ene-3-O-trimethylsilyl-1-thio-alpha-D-mannopyranoside was adopted for this particular purpose, which afforded beta-mannoside p-methoxyphenyl 2,3-O-(p-methoxybenzylidene)-4,6-O-(1,1,3,3-tetraisopropyl)+ ++disiloxanylidene-beta-D-mannopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside stereoselectively in 75% yield.     

Structural Characterization of a Gcn5-Related N-Acetyltransferase from Staphylococcus aureus

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphyloccocus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.     

Identification and biosynthesis of cyclic enterobacterial common antigen in Escherichia coli

Phosphoglyceride-linked enterobacterial common antigen (ECA(PG)) is a cell surface glycolipid that is synthesized by all gram-negative enteric bacteria. The carbohydrate portion of ECA(PG) consists of linear heteropolysaccharide chains comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The potential reducing terminal GlcNAc residue of each polysaccharide chain is linked via phosphodiester linkage to a phosphoglyceride aglycone. We demonstrate here the occurrence of a water-soluble cyclic form of enterobacterial common antigen, ECA(CYC), purified from Escherichia coli strains B and K-12 with solution nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and additional biochemical methods. The ECA(CYC) molecules lacked an aglycone and contained four trisaccharide repeat units that were nonstoichiometrically substituted with up to four O-acetyl groups. ECA(CYC) was not detected in mutant strains that possessed null mutations in the wecA, wecF, and wecG genes of the wec gene cluster. These observations corroborate the structural data obtained by NMR and ESI-MS analyses and show for the first time that the trisaccharide repeat units of ECA(CYC) and ECA(PG) are assembled by a common biosynthetic pathway.     

Pyruvated galactose and oligosaccharides from Erwinia chrysanthemi Ech6 extracellular polysaccharide

The acidic extracellular polysaccharide of Ech6 was depolymerized by fuming HCl. The pyruvated sugars were isolated and characterized by methods that included a combination of low-pressure gel-filtration and high-pH anion-exchange chromatographies, methylation linkage analyses, mass (GC-MS and MALDI-TOF MS) and 1H NMR (1D and 2D) spectroscopies. The following pyruvated sugars were obtained: 4,6-O-(1-carboxyethylidene)-D-Galp; 4,6-O-(1-carboxyethylidene)- alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-D-Galp; 4,6-O-(1-carboxyethylidene)-alpha-D-Galp-(1-->4)-alpha-D-GlcAp- (1-->3)-alpha-D-Galp-(1-->3)-L-Fucp; 4,6-O-(1-carboxyethylidene)-alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3) -alpha-D-Galp-(1-->3)-L-[beta-D-Glcp-(1-->4)]-Fucp. These oligosaccharides present potential haptenes for the development of specific antibodies and confirm the partial structure proposed previously for the extracellular polysaccharide from Erwinia chrysanthemi Ech6 [Yang, B. Y.; Gray, J. S. S.; Montgomery, R. Int. J. Biol. Macromol., 1994, 16, 306-312].     

Structure of the O-polysaccharide of Idiomarina zobellii KMM 231T containing two unusual amino sugars with the free amino group, 4-amino-4,6-dideoxy-D-glucose and 2-amino-2-deoxy-L-guluronic acid

Mild acid degradation of the lipopolysaccharide of the bacterium Idiomarina zobellii, type strain KMM 231T, with aq 2% HOAc at 100 degrees C, yielded an oligosaccharide, which represents one repeating unit of the O-polysaccharide. A polysaccharide was obtained by mild base degradation of the lipopolysaccharide. The following structure of the O-polysaccharide was elucidated by 1H and 13C NMR spectroscopy of the oligosaccharide and base-degraded lipopolysaccharide, including COSY, TOCSY, ROESY, 1H, 13C HSQC, HSQC-TOCSY and HMBC experiments: [-->3)-alpha-D-Quip4N-(1-->4)-alpha-D-GlcpA-(1-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-L-GulpNA-(1-->3)-beta-D-FucpNAc-(1-->] The O-polysaccharide is distinguished by the presence of two unusual amino sugars, 4-amino-4,6-dideoxy-D-glucose (D-Qui4N) and 2-amino-2-deoxy-L-guluronic acid (L-GulNA), both having the free amino group. The unexpectedly high acid lability of the glycosidic linkage of 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) could be associated with the presence of a free amino group adjacent to the site of attachment of FucNAc to Qui4N.     

Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis

The polysaccharide chains of enterobacterial common antigen (ECA) consist of linear trisaccharide repeat units with the structure -->3)- alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1--> 4)-alpha-d-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido-4, 6-dideoxy-d-galactose, ManNAcA is N - acetyl-d- mannosaminuronic acid, and GlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phospholipid moiety functions to anchor molecules in the outer membrane. The ECA trisaccharide repeat unit is assembled as a polyisoprenyl-linked intermediate which has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc- pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a block-polymerization mechanism. However, the identity of the polyisoprenoid carrier-lipid has not been established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier involved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNAcA-GlcNAc- pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol, undecaprenol. These analyses also resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be 1,2-diacyl- sn -glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP- disaccharide). An examination of purified ECAPGby phosphorus-31 nuclear magnetic resonance spectroscopy confirmed that the polysaccharide chains are linked to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does not appear to be an intermediate in ECAPGsynthesis. Nevertheless, although the available evidence clearly indicate that lipid II is a precursor of DGP-disaccharide, the function of this novel glycolipid is not yet known, and it may be an intermediate in the biosynthesis of a molecule other than ECAPG.      

Synthesis of cis-(1-->3)-glycosides of allyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-glucopyranoside

Syntheses of allyl 2,3,4-tri-O-benzyl-alpha-D-gluco- and D-galactopyranosyluronate-(1-->3)-2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-glucopyranoside via oxidation of the hydroxymethyl group of allyl 2,3,4-tri-O-benzyl-alpha-D-gluco- and D-galactopyranosyl-(1-->3)-2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-glucopyranoside under Jones conditions are described. Structures of the title compounds were confirmed by (1)H and (13)C NMR spectroscopy.     

Synthesis of spacer-equipped phosphorylated di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139

The synthesis of oligosaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139 containing a 4,6-cyclic phosphate galactose residue linked to GlcNAc is described. 8-Azido-3,6-dioxaoctyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, obtained by condensation of 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide and 8-azido-3,6-dioxaoctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, was converted to 8-azido-3,6-dioxaoctyl 3-O-benzyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-6-O-benzyl-2-deoxy-beta-D-glucopyranoside (6) by reductive opening of the acetal, followed by deacetylation and selective benzylation. Phosphorylation of 6 furnished two isomeric 4,6-cyclic 2,2,2-trichloroethyl phosphates. Glycosylation of the (S)-phosphate with 2,4-di-O-benzyl-3,6-dideoxy-alpha-L-xylo-hexopyranosyl bromide under halide-assisted conditions gave the desired tetrasaccharide, together with a trisaccharide. Global deprotection and reduction of the azide to an amine was effected by catalytic hydrogenation/hydrogenolysis to give the deprotected tetrasaccharide, which is functionalized for conjugation.     

Reverse hydrolysis reaction of chitin deacetylase and enzymatic synthesis of beta-D-GlcNAc-(1-->4)-GlcN from chitobiose

We found that a chitin deacetylase from Colletotrichum lindemuthianum could acetylate free amino sugar residues into N-acetylated forms in the presence of 3.0 M sodium acetate. The result was analyzed using a beta-N-acetyl-hexosaminidase-coupled assay system with p-nitrophenyl 2-amino-2-deoxy-beta-D-glucopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta- D-glucopyranoside as the substrate, and the liberation of p-nitrophenol was observed as a consequence of enzymatic N-acetylation of the glucosamine residue at the nonreducing end of the substrate. The chitin deacetylase also acetylated chitobiose and chitotetraose as substrates, which was evidenced by the decrease in the amount of free amino sugar residues in the chitooligosaccharides. The reaction product of chitobiose after the acetylation reaction was exclusively 2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->4)-2-amino-2-deoxy-D-gluc ose [GlcNAcGlcN], the structure of which was determined by FABMS and NMR analyses. This study offers a novel method for enzymatic N-acetylation of amino sugars, and especially with chitobiose as substrate, a selectively N-acetylated product, GlcNAcGlcN, can be synthesized.     

Structural elucidation of polysaccharide part of glycoconjugate from Treponema medium ATCC 700293.

Glycoconjugates are distributed on the cell surfaces of some small-sized treponemes and have been reported to be completely different from lipopolysaccharides. We separated a glycoconjugate fraction from Treponema medium ATCC 700293, a medium-sized oral spirochete, to assess its immunobiological activities and elucidate the chemical structure of its polysaccharide part using phenol/water extraction, hydrophobic chromatography, and gel filtration. The glycoconjugate showed negligible or weak endotoxic and immunobiological properties. The chemical structure of the polysaccharide part was shown by two-dimensional NMR and MALDI-TOF-MS to be a tetrasaccharide backbone with two amino acids: [-->4)beta-d-GlcpNAc3NAcA(1-->4)beta-d-ManpNAc3NAOrn(1-->3)beta-d-GlcpNAc(1-->3)alpha-D-Fucp4NAsp(1-->] where GlcNAc3NAcA is 2,3-diacetamido-2,3-dideoxyglucuronic acid, ManNAc3NAOrn is Ndelta-(2-acetamido-3-amino-2,3-dideoxymannuronyl)ornithine, and Fuc4NAsp is 4-(alpha-aspartyl)amino-4,6-dideoxygalactose.     

Synthesis and conformational analysis of the repeating units of bacterial spore peptidoglycan

Deprotection of the fully blocked disacharide allyl O-(2-amino-4,6-O-benzylidene-3-O-[(R)-1-carboxyethyl]-2-deoxy-beta-D-glucopyranosyl-1',2-lactam)-(1-->4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside by selective de-O-allylation and parallel removal of the benzylidene and O-benzyl groups is described. The resulting beta-muramyl lactam-(1-->4)-GlcNAc disaccharide is characterised as the per-O-acetylated derivative by 1H and 13C NMR spectroscopy and X-ray structure analysis. Conformational analysis about glycosidic bond of repeating units of bacterial spore cortex is based on experimental data and molecular modelling.     

Structure of the phenol-soluble polysaccharide from Shewanella putrefaciens strain A6

The structure of the phenol-soluble polysaccharide from Shewanella putrefaciens strain A6 has been elucidated. Chemical modifications of the polymer in conjunction with 1H and 13C NMR spectroscopy, including 2D techniques, were employed in the analysis. It is concluded that the repeating unit is composed of two nine-carbon sugars as follows: -->4)-alpha-NonpA-(2-->3)-beta-Sugp-(1--> where alpha-NonpA is 5-acetamido-7-acetamidino-8-O-acetyl-3,5,7,9-tetradeoxy-L-glycero-alpha-D-galacto-non-2-ulosonic acid (8eLeg) and beta-Sugp is 2-acetamido-2,6-dideoxy-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-beta-D-galactopyranose, with the proposed name Shewanellose (She).     

Accumulation of a lipid-linked intermediate involved in enterobacterial common antigen synthesis in Salmonella typhimurium mutants lacking dTDP-glucose pyrophosphorylase.

The heteropolysaccharide chains of enterobacterial common antigen (ECA) are composed of linear trisaccharide repeat units having the structure----3)-alpha-Fuc4NAc-(1----4)-beta-D-ManNAcA-(1---- 4)-alpha-D-GlcNAc- (1----. Mutants of Salmonella typhimurium lacking the structural gene for dTDP-glucose pyrophosphorylase (rfbA) are severely impaired in their ability to synthesize dTDP-glucose, which is a precursor of dTDP-4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc), the donor of Fuc4NAc residues for ECA synthesis. These mutants synthesize only trace amounts of ECA, and they are hypersensitive to sodium dodecyl sulfate (SDS). Incubation of delta rfbA mutants with [3H]N-acetylglucosamine ([3H]GlcNAc) resulted in the accumulation of radioactivity in N-acetyl-D-mannosaminuronic acid (ManNAcA)-GlcNAc-pyrophosphorylundecaprenol (lipid II), the putative acceptor of Fuc4NAc residues in ECA synthesis. Lipid II did not accumulate in either wild-type cells or in rff mutants unable to synthesize ManNAcA. Both the accumulation of lipid II and the synthesis of trace amounts of ECA were abolished when delta rfbA mutants were grown in the presence of the antibiotic tunicamycin. Tunicamycin also prevented the SDS-mediated lysis of the mutants. SDS-resistant derivatives of delta rfbA mutants were isolated that were no longer able to synthesize trace amounts of ECA. Characterization of these derivatives revealed that they were defective in various steps of ECA synthesis leading to the synthesis of lipid II. The data support the conclusion that accumulation of lipid II is responsible in some way for the hypersensitivity of delta rfbA mutants to SDS.     

Crystal structure of the histone acetyltransferase Hpa2: A tetrameric member of the Gcn5-related N-acetyltransferase superfamily

We report the crystal structure of the yeast protein Hpa2 in complex with acetyl coenzyme A (AcCoA) at 2.4 A resolution and without cofactor at 2.9 A resolution. Hpa2 is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, a family of enzymes with diverse substrates including histones, other proteins, arylalkylamines and aminoglycosides. In vitro, Hpa2 is able to acetylate specific lysine residues of histones H3 and H4 with a preference for Lys14 of histone H3. Hpa2 forms a stable dimer in solution and forms a tetramer upon binding AcCoA. The crystal structure reveals that the Hpa2 tetramer is stabilized by base-pair interactions between the adenine moieties of the bound AcCoA molecules. These base-pairs represent a novel method of stabilizing an oligomeric protein structure. Comparison of the structure of Hpa2 with those of other GNAT superfamily members illustrates a remarkably conserved fold of the catalytic domain of the GNAT family even though members of this family share low levels of sequence homology. This comparison has allowed us to better define the borders of the four sequence motifs that characterize the GNAT family, including a motif that is not discernable in histone acetyltransferases by sequence comparison alone. We discuss implications of the Hpa2 structure for the catalytic mechanism of the GNAT enzymes and the opportunity for multiple histone tail modification created by the tetrameric Hpa2 structure.     

Synthesis of aminoethylglycosides with oligosaccharide GM1 and asialo-GM1 ganglioside chains

4'-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O- benzyl-6-O-benzoyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-1-thio-2-trichloroacetamido-beta-D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->3)- (4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-beta-D-galactopyranosyl)- (1-->4)-(2,3-di-O-benzyl-6-O-benzoyl-beta-D-galactopyranosyl)- (1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of beta-D-Gal- (1-->3)-beta-D-GalNAc-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc-OCH2CH2NH2 containing the oligosaccharide chain of asialo-GM1 ganglioside in 72% overall yield. Selective 3'-O-glycosylation of 2-azidoethyl 2,3,6-tri-O- benzyl-4-O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O- acetyl-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoroacetic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl- 3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and tri-fluoracetic acid afforded 2-azidoethyl[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl- 3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl) (2,6-di-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D- glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM3 ganglioside, in 79% yield. Its 4'-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O- acetyl-beta-D-galactopyranosyl) 1-thio-2-trichloroacetamido-beta-D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoroacetic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)- (1-->3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-beta-D- galactopyranosyl)-(1-->4)-[[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D- galacto-2-nonulopyranosyl)onate]-(2-->3)]-(2,6-di-O-benzyl-beta-D- galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of beta-D-Gal-(1-->3)-beta-D-GalNAc-(1-->4)-[alpha-D-Neu5Ac-(2-->3)]- beta-D-Gal-(1-->4)-beta-D-Glc-OCH2CH2NH2 containing the oligosaccharide chain of GM1 ganglioside. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

Synthesis of the pentasaccharide related to the repeating unit of the antigen from Shigella dysenteriae type 4 in the form of its methyl ester 2-(trimethylsilyl)ethyl glycoside

Starting from D-mannose, D-glucose and L-fucose, the pentasaccharide derivative methyl 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl-(1-->3)-2-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranosyl-(1-->3)-2-O-acetyl-6-O-benzyl-4-O-(2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl)-alpha-D-mannopyranosyl-(1-->4)-[2-(trimethylsilyl)ethyl 2,3-di-O-benzyl-beta-D-glucopyranosid]uronate was synthesized. This compound with two alpha-mannopyranosyl units was transformed, via Walden inversion and subsequent deprotection, into the alpha-D-glucosamine-type target compound, namely methyl alpha-L-fucopyranosyl-(1-->3)-2-acetamido-2-deoxy-alpha-D-glucopyranosyl-(1-->3)-2-acetamido-2-deoxy-4-O-(alpha-L-fucopyranosyl)-alpha-D-glucopyranosyl-(1-->4)-[2-(trimethylsilyl)ethyl beta-D-glucopyranosid]uronate which is related to the repeating unit of the O-antigen from Shigella dysenteriae type 4.     

Synthesis of N-[2-O-(2-acetamido-2,3-dideoxy-5-thio-d-glucopyranose-3-yl)-d-lactoyl]-l-alanyl-d-isoglutamine

N-[2-O-(2-Acetamido-2,3-dideoxy-5-thio-d-glucopyranose-3-yl)-d-lactoyl]-l-alanyl-d-isoglutamine, in which the ring-oxygen atom of the sugar moiety in N-acetylmuramoyl-l-alanyl-d-isoglutamine (MDP) has been replaced by sulfur, was synthesized from 2-acetamido-2-deoxy-5-thio-α-d-glucopyranose (1). O-Deacetylation of the acetylated acetal, derived from the methyl α-glycoside of 1 by 4,6-O-isopropylidenation and subsequent acetylation, gave methyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-5-thio-α-d-glucopyranoside (4). Condensation of 4 with l-2-chloropropanoic acid, and subsequent esterification, afforded the corresponding ester, which was converted, viaO-deisopropylidenation, acetylation, and acetolysis, into 2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-5-thio-α-d-glucopyranose (12). Coupling of the acid, formed from 12 by hydrolysis, with the methyl ester of l-alanyl-d-isoglutamine, and de-esterification, yielded the title compound.