Toshiki Mine

An α2,6-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. The truncated recombinant enzyme was purified as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis through 3 column chromatography steps. The enzyme had distinct activity compared with known marine bacterial alpha2,6-sialyltransferases. Although alpha2,6-sialyltransferases cloned from marine bacteria, such as Photobacterium damselae strain JT0160, P. leiognathi strain JT-SHIZ-145, and Photobacterium sp. strain JT-ISH-224, show only alpha2,6-sialyltransferase activity, the recombinant enzyme cloned from P. leiognathi strain JT-SHIZ-119 showed both alpha2,6-sialyltransferase and alpha2,6-linkage-specific neuraminidase activity. Our results provide important information toward a comprehensive understanding of the bacterial sialyltransferases belonging to the group 80 glycosyltransferase family in the CAZy database.

An α2,3-Sialyltransferase from Photobacterium sp. JT-ISH-224 Transfers N-Acetylneuraminic Acid to Both the O-2 and O-3′ Hydroxyl Groups of Lactose

We found that α2,3-sialyltransferase from Photobacterium sp. JT-ISH-224 produced a regio-mistaken sialyl-transferred by-product. Spectroscopic analysis of the purified by-product indicated that it contained two N-acetylneuraminic acids: one attached to the O-3′ hydroxyl group of lactose, and the other attached to the O-2 hydroxyl group of lactose. The relative configuration between the C-1 and C-3 of the α-glucopyranose residue is superimposable with that between C-4 and C-2 of galactopyranoside. Therefore, formation of this by-product, designated 2,3′-disialyllactose, was simply rationalized as a regio-mistaken reaction of bacterial α2,3-sialyltransferase. This finding indicates that this bacterial α2,3-sialyltransferase has a possibility to synthesize several unusual sialosides.

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. phosphoreum JT-ISH-467 as a model enzyme to study the role of the amino acid residues around the substrate-binding site. It was found that a mutation of the glutamic acid at position 342 in the sialyltransferase resulted in a loss of sialidase activity, although the mutant showed no decrease in sialyltransferase activity. Based on this result, it is strongly expected that the Glu342 of the enzyme is an important amino acid residue for sialidase activity.

Efficiency of organic solvents on the ability of α2,3-sialyltransferase from Photobacterium sp. JT-ISH-224 to control a hydrolysis side reaction

Enzymatic synthesis of oligosaccharides using specific sialyltransferases enables single-step glycosylation with high positional and anomeric structural selectivity. The α2,3-sialyltransferase cloned from the marine bacterium Photobacterium sp. JT-ISH-224 has unique and broad acceptor specificity, but this enzyme possesses not only sialyltransferase activity but also sialidase activity. To synthesize sialoside derivative effectively, only sialyltransferase activity is required. We report here that addition of organic solvents was effective to control the sialidase activity and a resulting product was not hydrolyzed. The enzyme was even active in the presence of acetonitrile, ethanol, methanol, or acetone. To determine the suitable concentrations of these organic solvents, only sialyltransferase activity could be allowed, and as a result, the stable synthesis of sialoside could be achieved.

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. The ganglioside generated by the catalytic activity of α2-3ST was identified as GM1b. On the other hand, when enzyme reactions by α2-6STs were performed using substrates GA2 and GA1, very unique gangliosides were generated. The structures were identified as NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer and NeuAcα2-6Galβ1-3GalNAcβ1-4Galβ1-4Glcβ-Cer, respectively. The synthesized ganglioside NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer showed binding activity to the influenza A virus {A/Panama/2007/99 (H3N2)} at a similar level to purified sialyl(α2-3)paragloboside (S2-3PG) and sialyl(α2-6)paragloboside (S2-6PG) from mammalian sources. The evidence suggests that these STs have unique features, including substrate specificities restricted not only to lacto-series but also to ganglio-series GSLs, as well as catalytic potentials for ganglioside synthesis. This evidence demonstrates that effective in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing sialic acid (Sia) modifications, thereby preparing large-scale gangliosides and permitting the exploration of unknown functions.

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. There have been no previous reports of marine bacteria producing fucosyltransferase. This paper thus represents the first report of fucosyltransferase-producing marine bacteria.

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. The activity of the enzyme was maximal at 35 °C at pH 9.0, and the synthetase required Mg2+ for activity. Although these properties are similar to those of other CMP-Neu5Ac synthetases isolated from bacteria, this synthetase produced not only CMP-Neu5Ac from cytidine triphosphate and Neu5Ac, but also CMP-N-glycolylneuraminic acid from cytidine triphosphate and N-glycolylneuraminic acid, unlike CMP-Neu5Ac synthetase purified from Escherichia coli.

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. JT-ISH-224 and TM2 were connected via a peptide linker (ST-L-TM2) was constructed and expressed in Escherichia coli. The ST-L-TM2 was produced in the soluble form with a yield of approximately 15,000 unit/300 ml of the E. coli culture. The ST-L-TM2 was partially purified and part of it was immobilized onto biotin-bearing magnetic microbeads. The immobilized ST-L-TM2 onto microbeads could be used at least seven consecutive reaction cycles with no observed decrease in enzymatic activity. In addition, the optimum pH and temperature of the immobilized enzyme were changed compared to those of a free form of the ST. Considering these results, it was strongly expected that the immobilized ST-L-TM2 was a promising tool for the production of various kind of sialoligosaccharides.

Transfructosylation reaction in cured tobacco leaf (Nicotiana tabacum)

Tobacco plant was known to be a non-fructan-storing plant. However, we demonstrated that fructo-oligosaccharides (FOSs) were formed in cured tobacco leaf on adding sucrose to the leaf in our previous report (Nagai et al., J. Agric. Food Chem., 60, 6606-6612, 2012). Also, it was expected from the results obtained in previous study that FOSs were generated by enzymatic reaction in cured tobacco leaf. The purpose of this study is to confirm and understand the mechanisms of above-mentioned FOSs formation. Thus, we tried to purify the enzymes related to the production of FOSs. The enzymes were extracted from pulverized cured tobacco leaf (burley type leaf), and were purified by charcoal treatment, ultrafiltration, and several chromatography techniques. As a result, one of the enzymes was purified up to 414-fold. It was revealed that this enzyme was acid invertase exhibiting maximum transfructosylation activity at pH 6.0, 60 °C. In addition, general properties of this enzyme were also investigated. The enzyme purified in this study enhanced the ratio of FOSs formation under the condition of high concentrated sucrose. From these results, it was suggested that this enzyme participated in the formation of FOSs in tobacco leaf after curing.

Purification of Marine Bacterial Sialyltransferases and Sialyloligosaccharides

Sialic acids are important components of carbohydrate chains and are usually found at the terminal position of the carbohydrate moiety of glycoconjugates (Angata & Varki, 2002; Schauer, 2004). Sialyloligosaccharides of glycoconjugates play important roles in many biological processes (Gagneux & Varki, 1999; Varki, 1993). The transfer of sialic acids to carbohydrate chains is performed by specific sialyltransferases in the cell (Angata & Varki, 2002; Vimr et al., 2004). Thus, sialyltransferases are considered to be key enzymes in the biosynthesis of sialylated glycoconjugates. Detailed investigations of the biological functions of sialylated glycoconjugates require an abundant supply of the target compounds. To date, many sialyltransferases, and the genes encoding them, have been isolated from various sources including mammalian, bacterial, and viral sources (Schauer, 2004; Sujino et al., 2000; Yamamoto et al., 2006). During our research, we have isolated over 20 bacteria that produce sialyltransferase and have revealed the characteristics of these enzymes (Kajiwara et al., 2009; Yamamoto, 2010). In this chapter, we will introduce our research activities focusing on methods for (1) screening bacteria for glycosyltransferase activity; (2) purifying native sialyltransferases from marine bacteria; and (3) synthesizing and purifying sialyloligosaccharides produced by marine bacterial sialyltransferases.