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Journal of Biochemistry Advance Access published online on November 4, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm208
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© 2007 The Japanese Biochemical Society

Photobacterium sp. JT-ISH-224 produces two sialyltransferases, {alpha}-/ß-galactoside {alpha}2,3-sialyltransferase and ß-galactoside {alpha}2,6-sialyltransferase

Hiroshi Tsukamoto1, Yoshimitsu Takakura, Toshiki Mine and Takeshi Yamamoto

Glycotechnology Business Unit, Japan Tobacco Inc., Higashibara, Iwata, Shizuoka 438-0802, JAPAN

1To whom correspondence should be addressed; Dr. Hiroshi Tsukamoto, Tel.: +81-538-32-7389; FAX: +81-538-33-6046; E-mail: takeshi.yamamoto{at}ims.jti.co.jp; or Tel.: +81-538-32-7337; FAX: +81-538-33-6046; hiroshi.tsukamoto{at}ims.jti.co.jp

Received August 21, 2007; Accepted October 22, 2007


  Abstract

A novel bacterium, Photobacterium sp. JT-ISH-224, that produces {alpha}-/ß-galactoside {alpha}2,3-sialyltransferase and ß-galactoside {alpha}2,6-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}-/ß-galactoside {alpha}2,3-sialyltransferase from P. phosphoreum and ß-galactoside {alpha}2,6-sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1230 and 1545 bp for encoding an {alpha}2,3-sialyltransferase and an {alpha}2,6-sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The {alpha}2,3-sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum {alpha}2,3-sialyltransferase, whereas the {alpha}2,6-sialyltransferase had 54% amino acid sequence identity with the P. damselae {alpha}2,6-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 {alpha}2,3-sialyltransferase catalyzed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, {alpha}-methyl-galactopyranoside and ß-methyl-galactopyranoside with low apparent Km and the {alpha}2,6-sialyltransferase catalyzed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent Km.

Key Words: bacterial sialyltransferase, glycosyltransferase family 80, Photobacterium, sialic acid


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