Immobilized sialyltransferase fused to a fungal biotin-binding protein: Production, properties, and applications.

A β-galactoside α2,6-sialyltransferase (ST) from the marine bacterium Photobacterium sp. JT-ISH-224 with a broad acceptor substrate specificity was fused to a fungal biotin-binding protein tamavidin 2 (TM2) to produce immobilized enzyme. Specifically, a gene for the fusion protein, in which ST from Photobacterium sp.

Unique gangliosides synthesized in vitro by sialyltransferases from marine bacteria and their characterization: ganglioside synthesis by bacterial sialyltransferases.

On the basis of the results outlined in our previous report, bacterial sialyltransferases (ST) from marine sources were further characterized using glycosphingolipids (GSL), especially ganglio-series GSLs, based on the enzymatic characteristics and kinetic parameters obtained by Line weaver-Burk plots. Among them, GA1 and GA2 were found to be good substrates for these unique STs. Thus, new gangliosides synthesized by α2-3 and α2-6STs were structurally characterized by several analytical procedures.

Isolation of fucosyltransferase-producing bacteria from marine environments.

Fucose-containing oligosaccharides on the cell surface of some pathogenic bacteria are thought to be important for host-microbe interactions and to play a major role in the pathogenicity of bacterial pathogens. Here, we screened marine bacteria for glycosyltransferases using two methods: a one-pot glycosyltransferase assay method and a lectin-staining method. Using this approach, we isolated marine bacteria with fucosyltransferase activity.

Loss-of-function mutation in bi-functional marine bacterial sialyltransferase.

An α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a bi-functional enzyme showing both α2,3-sialyltransferase and α2,3-linkage specific sialidase activity. To date, the crystal structures of several sialyltransferases have been solved, but the roles of amino acid residues around the catalytic site have not been completely clarified. Hence we performed a mutational study using α2,3-sialyltransferase cloned from P.

Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties.

The avidin-biotin interaction is widely employed as a universal tool in numerous biotechnological applications. In avidin-biotin technology, non-specific binding to biological macromolecules is a hindrance. The major origin of this non-specific binding is the electrical charge of the surface of biotin-binding proteins.

A CMP-N-acetylneuraminic acid synthetase purified from a marine bacterium, Photobacterium leiognathi JT-SHIZ-145.

A cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase was found in a crude extract prepared from Photobacterium leiognathi JT-SHIZ-145, a marine bacterium that also produces a β-galactoside α2,6-sialyltransferase. The CMP-Neu5Ac synthetase was purified from the crude extract of the cells by a combination of anion-exchange and gel filtration column chromatography. The purified enzyme migrated as a single band (60 kDa) on sodium dodecylsulfate-polyacrylamide gel electrophoresis.

A recombinant α-(2→3)-sialyltransferase with an extremely broad acceptor substrate specificity from Photobacterium sp. JT-ISH-224 can transfer N-acetylneuraminic acid to inositols.

We confirmed that a recombinant α-(2→3)-sialyltransferase cloned from Photobacterium sp. JT-ISH-224 recognizes inositols having a structure corresponding to the C-3 and C-4 of a galactopyranoside moiety, such as epi-, 1d-chiro, myo-, and muco-inositol, as acceptor substrates, and that the enzyme can transfer N-acetylneuraminic acid (Neu5Ac) from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) to them. After purifying the reaction products, the structures were confirmed by use of NMR spectroscopy and mass spectrometry.

Enzymatic synthesis of unique sialyloligosaccharides using marine bacterial alpha-(2-->3)- and alpha-(2-->6)-sialyltransferases.

We investigated the acceptor substrate specificities of marine bacterial alpha-(2-->3)-sialyltransferase cloned from Photobacterium sp. JT-ISH-224 and alpha-(2-->6)-sialyltransferase cloned from Photobacterium damselae JT0160 using several saccharides as acceptor substrates. After purifying the enzymatic reaction products, we confirmed their structure by NMR spectroscopy.

Efficient synthesis of 6'-sialyllactose, 6,6'-disialyllactose, and 6'-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224.

We have previously reported the efficient conversion of lactose into 3'-sialyllactose by high cell density cultures of a genetically engineered Escherichia coli strain expressing the Neisseria meningitidis gene for alpha-(2-->3)-sialyltransferase [Fierfort, N.; Samain, E. J.

Visualization of sialic acid produced on bacterial cell surfaces by lectin staining.

Oligosaccharides containing N-acetylneuraminic acid on the cell surface of some pathogenic bacteria are important for host-microbe interactions. N-acetylneuraminic acid (Neu5Ac) plays a major role in the pathogenicity of bacterial pathogens. For example, cell surface sialyloligosaccharide moieties of the human pathogen Haemophilus influenzae are involved in virulence and adhesion to host cells.

Tamavidin, a versatile affinity tag for protein purification and immobilization.

Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity and is highly produced in soluble form in Escherichia coli. By contrast, widely used biotin-binding proteins avidin and streptavidin are rarely produced in soluble form in E. coli.

Sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates.

Bacterial sialyltransferases (STs) from marine sources were characterized using glycosphingolipids (GSLs). Bacterial STs were found to be beta-galacotoside STs. There were two types of STs: (1) ST obtained from strains such as ishi-224, 05JTC1 (#1), ishi-467, 05JTD2 (#2), and faj-16, 05JTE1 (#3), which form alpha2-3 sialic acid (Sia) linkages, named alpha2-3ST, (2) ST obtained from strains such as ISH-224, N1C0 (#4), pda-rec, 05JTB2 (#5), and pda-0160, 05JTA2 (#6), which form alpha2-6 Sia linkages, named alpha2-6ST.

An alpha2,6-sialyltransferase cloned from Photobacterium leiognathi strain JT-SHIZ-119 shows both sialyltransferase and neuraminidase activity.

We cloned, expressed, and characterized a novel beta-galactoside alpha2,6-sialyltransferase from Photobacterium leiognathi strain JT-SHIZ-119. The protein showed 56-96% identity to the marine bacterial alpha2,6-sialyltransferases classified into glycosyltransferase family 80. The sialyltransferase activity of the N-terminal truncated form of the recombinant enzyme was 1477 U/L of Escherichia coli culture.

Crystal structure of alpha/beta-galactoside alpha2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum.

Alpha/beta-galactoside alpha2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both alpha-galactoside and beta-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the alpha2,3-sialyltransferase in complex with CMP.

Crystal structure of Vibrionaceae Photobacterium sp. JT-ISH-224 alpha2,6-sialyltransferase in a ternary complex with donor product CMP and acceptor substrate lactose: catalytic mechanism and substrate recognition.

Sialyltransferases are a family of glycosyltransferases that catalyze the transfer of N-acetylneuraminic acid residues from cytidine monophosphate N-acetylneuraminic acid (CMP-NeuAc) as a donor substrate to the carbohydrate groups of glycoproteins and glycolipids as acceptor substrates. We determined the crystal structure of Delta16psp26ST, the N-terminal truncated form of alpha2,6-sialyltransferase from Vibrionaceae Photobacterium sp. JT-ISH-224, complexed with a donor product CMP and an acceptor substrate lactose.

A beta-galactoside alpha2,6-sialyltransferase produced by a marine bacterium, Photobacterium leiognathi JT-SHIZ-145, is active at pH 8.

A gene encoding a sialyltransferase produced by Photobacterium leiognathi JT-SHIZ-145 was cloned, sequenced, and expressed in Escherichia coli. The sialyltransferase gene contained an open reading frame of 1494 base pairs (bp) encoding a predicted protein of 497 amino acid residues. The deduced amino acid sequence of the sialyltransferase had no significant similarity to mammalian sialyltransferases and did not contain sialyl motifs, but did show high homology to another marine bacterial sialyltransferase, a beta-galactoside alpha2,6-sialyltransferase produced by P.

Purification, crystallization and preliminary crystallographic characterization of the alpha 2,6-sialyltransferase from Photobacterium sp. JT-ISH-224.

Sialyltransferases transfer sialic acid from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the nonreducing termini of the oligosaccharyl structures of various glycoproteins and glycolipids. The newly cloned alpha2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 (from the Vibrionaceae family) is composed of two domains: an unknown N-terminal domain and a catalytic C-terminal domain which shares significant homology with the Pasteurella multocida multifunctional sialyltransferase.