Purification and Characterization of a Marine Bacterial  -Galactoside  2,6-Sialyltransferase from Photobacterium damsela JTO16O

Abstract

A bacterial sialyltransferase, named sialyltransferase 0160, was purified from a marine bacterium that had been isolated from seawater from Sagami Bay, Kanagawa. This strain has been identified as Photobacterium damsela, and named P. damsela JT0160. Sialyltransferase 0160 was purified 688-fold to homogeneity from the crude extract of the cells with a yield of 19% using a combination of anion exchange chromatography, hydroxyapatite chromatography, gel filtration chromatography, and affinity chromatography. The purified enzyme migrated as a single band (61 kDa) on sodium dodecyl sulfate-polyacrylamide gel. This sialyltransferase was found to be a beta-galactoside alpha 2,6-sialyltransferase [EC 2.4.99.1] which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6 on the basis of an analysis of the enzymatic reaction products with HPLC, 1H-, 13C-NMR spectroscopy, and fast atom bombardment mass spectroscopy.

Loss-of-Function Mutation in Bi-Functional Marine Bacterial Sialyltransferase

“…After the reaction, the reaction mixtures were analyzed by HPLC as previously described. 34) In the HPLC method with 3 0 -sialyllactose, the enzyme reaction mixture (150 mL) consisted of a sample of the enzyme, 100 mM 3 0 -sialyllactose, 100 mM Bis-Tris buffer (pH 6.0), and 0.5 M NaCl at 30 C. The reactions were stopped by heat treatment (100 C, 5 min). Then the reaction mixtures were immediately analyzed by HPLC, as previously described.…”

Purification, Cloning, and Expression of an α/β-Galactoside α-2,3-Sialyltransferase from a Luminous Marine Bacterium, Photobacterium phosphoreum

“…The apparent K m values for lactose (1.7 mM) and N-acetyllactosamine (2.5 mM) were lower than the apparent K m of the enzyme from P. damselae, and the apparent K m for monosaccharides was much lower (0.54 mM for methyl-␣-D-galactopyranoside and 1.3 mM for methyl-␤-D-galactopyranoside). The finding that the apparent K m for ␣-galactoside was lower than the apparent K m for ␤-galactoside is unprecedented, because the enzyme from P. damselae favors ␤-galactoside over ␣-galactoside (21), and the sialyltransferase from N. meningitidis has not been studied quantitatively (13). The apparent K m of the sialyltransferase from JT-ISH-467 for CMP-NeuAc (0.050 mM) is one of the lowest among apparent K m values of the bacterial sialyltransferases, which range from 0.014 mM (N. gonorrhoeae) (41), 0.32 mM (P. damselae) (21), and 0.44 mM (P. multocida subsp.…”

“…Bacterial sialyltransferases are less specific in this regard, and this property is considered useful when various sialylated glycans are to be prepared. For example, the sialyltransferase from P. damselae had high affinity to both lactose (apparent K m of 6.82 mM) and N-acetyllactosamine (apparent K m of 8.95 mM) and was even active for a monosaccharide, methyl-␤-D-galactopyranoside, with a somewhat higher apparent K m of 174 mM (21). The sialyltransferase from JT-ISH-467 was distinctive.…”

“…The reaction was carried out at 25 or 30°C. After the reaction, the reaction mixture was diluted with 5 mM potassium phosphate buffer (pH 6.8) to 2 ml and applied to a column (0.5 ϫ 2 cm) of AG1-X2 resin (Bio-Rad), which was equilibrated with 1 M potassium phosphate buffer (pH 6.8) (21). The eluate (2 ml) was collected and mixed with the scintillation mixture Ultima Gold from Packard.…”

Purification, crystallization and preliminary crystallographic characterization of the α2,6-sialyltransferase fromPhotobacteriumsp. JT-ISH-224

“…The protein determination was performed using a protein assay (Bio-Rad) with bovine serum albumin as a standard. The activity of 2,6-STase was measured according to the method of Yamamoto et al (1996).…”