T. N. Druzhinina

Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157

The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was β1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.

Biochemical characterization of a new β-1,3-galactosyltransferase WbuP from Escherichia coli O114 that catalyzes the second step in O-antigen repeating-unit.

In this study, synthetic acceptor substrate GlcNAc alpha-PO3-PO3-(CH2)11-O-phenyl (GlcNAc-PP-PhU) was employed in glycosyl transferase assays to characterize the WbuP galactosyltransferase activity. This activity was time- and enzyme concentration-dependent. The optimal enzyme activity was observed at pH 6.5 and 25°C. The enzyme requires Mn(2+) ions for maximal activity and detergents in the assay did not increase glycosyltransfer activity. The enzyme was shown to be specific for the UDP-Gal donor substrate. Kinetic parameters were determined for UDP-Gal, and GlcNAc-PP-PhU. The enzyme product was determined to have a β-1,3-linkage using strategies based on exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as well as collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MS(n)). Our results conclusively demonstrate that the wbuP gene of Escherichia coli O114 encodes a UDP-Gal: GlcNAc α-pyrophosphate-lipid β-1,3-Gal-transferase that transfers the second sugar moiety in the assembly of the O114 repeating unit.

Simple synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-α-D-galactopyranosyl) diphosphate

A simple method of the synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-α-D-galactopyranosyl) diphosphate, which is a synthetic lipid acceptor for glycosyl transferases participating in the biosynthesis of O-antigenic polysaccharides of Gram-negative bacteria, is suggested.

11-Phenoxyundecyl phosphate as a 2-acetamido-2-deoxy-α-d-glucopyranosyl phosphate acceptor in O-antigen repeating unit assembly of Salmonella arizonae O:59 ☆

A synthesis of 11-phenoxyundecyl phosphate and its biochemical transformation (using GlcNAc-P transferase from Salmonella arizonae O:59 membranes catalysing transfer of GlcNc-phosphate from UDP-GlcNAc on lipid-phosphate) into P(1)-11-phenoxyundecyl, P(2)-2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate are described.

Synthesis of 11-phenoxyundecyl phosphate and its use as a substrate-acceptor in the reaction with UDP-GlcNAc: polyprenyl phosphate GlcNAc-phosphotransferase from Salmonella arizona O:59

A new scheme of synthesis of 11-phenoxyundecyl phosphate from 11-bromoundecanoic acid was suggested; its ability to serve as an acceptor of 2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate in a reaction catalyzed by UDP-N-acetylglucosamine: polyprenyl phosphate N-acetylglucosamine phosphotransferase from Salmonella arizona O:59 was demonstrated.

Synthesis of P 1-(11-phenoxyundecyl)-P 2-(α-D-galactopyranosyl) diphosphate and P 1-(11-phenoxyundecyl)-P 2-(α-D-glucopyranosyl) diphosphate and investigation on their acceptor properties in the reaction of mannosyl residue transfer catalyzed by mannosyltransferase from Salmonella newport

P1-(11-phenoxyundecyl)-P2-(α-D-galactopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(α-D-glucopyranosyl) diphosphate have been synthesized for the first time, and their ability to serve as a mannosyl residue substrate-acceptors in the enzymatic reaction, catalyzed by mannosyltransferase membrane preparation from Salmonella newport cells, was investigated. It was demonstrated that the derivative containing galactopyranose residue is able to accept the mannosyl residue from GDP-Man, while the derivative containing glucopyranose residue does not have such an ability.

The synthesis of P 1-[11-(anthracen-9-ylmethoxy)undecyl]-P 2-(2-acetamido-2-deoxy-α-D-glucopyranosyl) diphosphate and the study of its acceptor properties in the enzymatic reaction catalyzed by a D-rhamnosyltransferase from Pseudomonas aeruginosa

P1-[11-(anthracen-9-ylmethoxy)undecyl]-P2-(2-acetamido-2-deoxy-α-D-glucopyranosyl) diphosphate, a fluorescent derivative of undecyl diphosphate 2-acetamido-2-deoxyglucose, was chemically synthesized. The ability of the compound to serve as acceptor substrate for the transfer of D-rhamnose residue by D-rhamnosyltransferase WbpZ from Pseudomonas aeruginosa PAO1 was demonstrated.

The synthesis of 11-[(2-pyridyl)amino]- and 11-[(9-anthracenylcarbonyl)amino]undecyl phosphate and the study of their acceptor properties in the enzymatic reaction catalyzed by Salmonella galactosyl phosphotransferases

Undecyl phosphate derivatives with new fluorescent labels, 11-[(2-pyridyl)amino]undecyl phosphate and 11-[(9-anthracenylcarbonyl)amino]undecyl phosphate, were synthesized. These compounds were shown to be acceptor substrates of the galactosyl phosphate residue in the enzymatic reaction catalyzed by galactosyl phosphotransferase from Salmonella anatum or Salmonella newport membrane preparations.

Synthesis and acceptor properties of 11-[(9′-Anthracenyl)methoxy]undecyl Phosphate and P 1 -{11-[(9′-anthracenyl)methoxy]undecyl}- P 2 -(α-D-galactopyranosyl) diphosphate in enzymatic reactions catalyzed with galactosylphosphotransferase and mannosyltransferase from Salmonella newport

Derivatives of undecyl phosphate containing the fluorescent label-11-[(9′-anthracenyl)methoxy]undecyl phosphate and P1-{11-[(9’-anthracenyl)methoxy]undecyl}-P2-(α-D-galactopyranosyl) diphosphate—were synthesized for the first time. An ability of the substituted undecyl phosphate to serve as an acceptor substrate of the galactosyl phosphate residue, and of the respective galactosyl diphosphate derivative as an acceptor substrate of the mannose residue in the reactions catalyzed with galactosylphosphotransferase and mannosyltransferase of the membrane preparation from Salmonella newport cells, respectively, was shown.

New fluorescent analogs of bacterial undecaprenyldiphosphate galactose

A synthesis of four new analogs of bacterial undecaprenyldiphosphate galactose, in which the oligoisoprenoid moiety is replaced with a linear hydrocarbon chain, was accomplished. These compounds contain a fluorescent anthracenyl group at the ω-end of the lipophilic chain variable in length. A comparative analysis of their ability to serve as substrates-acceptors of the mannose moiety in the enzymatic reactions catalyzed by mannosyltransferase from bacteria Salmonella newport cell membranes was performed.