Influenza B virus neuraminidase can synthesize its own inhibitor.

BACKGROUND: Neuraminidase, one of the two surface glycoproteins of influenza virus, cleaves terminal sialic acid residues from glycolipids or glycoproteins. Its crystal structure is known at high resolution, but the mechanism of glycosyl hydrolysis remains unclear. RESULTS: We have determined the crystal structure at 1.8 A resolution of two complexes of influenza B/Beijing neuraminidase containing either the reaction product, sialic acid, or the transition state analogue inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). The sialic acid is bound in a distorted 'boat' conformation closely resembling that of bound DANA, stabilized by a conserved tyrosine residue (Tyr408). This distortion also gives rise to a suicidal side reaction that converts sialic acid to DANA at a low rate. CONCLUSIONS: The mechanism of neuraminidase action is distinct from that of other known glycosyl hydrolases. Substrate distortion appears to be the driving force in glycosyl bond hydrolysis and the proton required for catalysis can probably be donated by water, rather than by residues in the active site, thus allowing the enzyme to operate at high pH. The side reaction converting sialic acid to DANA appears reasonably favourable, and it is unclear how this is minimized by the enzyme.

Chemoenzymatic synthesis of modified maltooligosaccharides from cyclodextrin derivatives.

Methyl and p-nitrophenyl alpha-maltooligosaccharides with a 3,6-anhydro ring on the fourth glucosyl residue, starting from the reducing end, were prepared. Enzymatic coupling catalyzed by CGTase, between 3A,6A-anhydrocyclomaltohexaose and methyl or p-nitrophenyl alpha-D-glucosides led to maltohepatosides. When miglitol, a nojirimycin analogue was used, maltooligosaccharides with miglitol at the reducing end were also obtained. After glucoamylase digestion, maltopentaosides with a 3,6-anhydro glucose as antepenultimate unit were produced in good yield. The same methyl maltopentaoside was also obtained when 3A,6A-anhydrocyclomaltoheptaose was incubated with methyl alpha-D-glucoside and CGTase, glucoamylase, glucose oxidase and catalase. These results provided new information about the specificity of the subsites of CGTase.

Structure of an extracellular polysaccharide produced by Lactobacillus rhamnosus strain C83.

The extracellular polysaccharide produced by Lactobacillus rhamnosus strain C83 was found to be composed of D-glucose and D-galactose in a molar ratio of 2:3. The primary structure of the polysaccharide was shown by sugar analysis, methylation analysis, FABMS, partial acid hydrolysis and nuclear magnetic resonance (NMR) spectroscopy to consist of a pentasaccharide repeating unit having the following structure: -->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6 )-alpha-D -Glcp-(1-->3)-beta-D-Galf-(1-->

In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacter pylori alpha-1,3 fucosyltransferase in engineered Escherichia coli.

We report here the in vivo production of type 2 fucosylated-N-acetyllactosamine oligosaccharides in Escherichia coli. Lacto-N-neofucopentaose Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc, lacto-N-neodifucohexaose Galbeta1-4(Fucalpha1-3)Glc-NAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc, and lacto-N-neodifucooctaose Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc were produced from lactose added in the culture medium. Two of them carry the Lewis X human antigen. High cell density cultivation allowed obtaining several grams of fucosylated oligosaccharides per liter of culture. The fucosylation reaction was catalyzed by an alpha-1,3 fucosyltransferase of Helicobacter pylori overexpressed in E. coli with the genes lgtAB of N. meningitidis. The strain was genetically engineered in order to provide GDP-fucose to the system, by genomic inactivation of gene wcaJ involved in colanic acid synthesis and overexpression of RcsA, positive regulator of the colanic acid operon. To prevent fucosylation at the glucosyl residue, lactulose Galbeta1-4Fru was assayed in replacement of lactose. Lactulose-derived oligosaccharides carrying fucose were synthesized and characterized. Fucosylation of the fructosyl residue was observed, indicating a poor acceptor specificity of the fucosyltransferase of H. pylori.

The living factory: in vivo production of N-acetyllactosamine containing carbohydrates in E. coli.

Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a beta(1,4)GlcNAc-oligosaccharide synthase), and the Neisseria beta(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as betaGal(1,4)[betaGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for betaGal(1,4)GlcNAc alpha(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.