Photobacterium sp. JT-ISH-224 produces two sialyltransferases, alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase.
A novel bacterium, Photobacterium sp. JT-ISH-224, that produces alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase, was isolated from the gut of a Japanese barracuda. The genes that encode the enzymes were cloned from the genomic library of the bacterium using the genes encoding alpha-/beta-galactoside alpha2,3-sialyltransferase from P. phosphoreum and beta-galactoside alpha2,6-sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1,230 and 1,545 bp for encoding an alpha2,3-sialyltransferase and an alpha2,6-sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The alpha2,3-sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum alpha2,3-sialyltransferase, whereas the alpha2,6-sialyltransferase had 54% amino acid sequence identity with the P. damselae alpha2,6-sialyltransferase. For both enzymes, the DNA fragments that encoded the full-length protein and its truncated form lacking the putative signal peptide sequence were amplified by a polymerase chain reaction and cloned into an expression vector. Each gene was expressed in Escherichia coli, and the lysate from each strain had enzymatic activity. The alpha2,3-sialyltransferase catalysed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside with low apparent K(m) and the alpha2,6-sialyltransferase catalysed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent K(m).
Tsukamoto
Hiroshi
H
Glycotechnology Business Unit, Japan Tobacco Inc., Higashibara, Iwata, Shizuoka, Japan. hiroshi.tsukamoto@ims.jti.co.jp
Takakura
Yoshimitsu
Y
Mine
Toshiki
T
Yamamoto
Takeshi
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010776:Photobacterium / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D017931:DNA Primers; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase