Purification, cloning, and expression of an alpha/beta-galactoside alpha-2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum.
A novel sialyltransferase, alpha/beta-galactoside alpha-2,3-sialyltransferase, was purified from the cell lysate of a luminous marine bacterium, Photobacterium phosphoreum JT-ISH-467, isolated from the Japanese common squid (Todarodes pacificus). The gene encoding the enzyme was cloned from the genomic library of the bacterium using probes derived from the NH(2)-terminal and internal amino acid sequences. An open reading frame of 409 amino acids was identified, and the sequence had 32% identity with that of beta-galactoside alpha-2,6-sialyltrasferase in Photobacterium damselae JT0160. DNA fragments that encoded the full-length protein and a protein that lacked the sequence between the 2nd and 24th residues at the NH(2) terminus were amplified by polymerase chain reactions and cloned into an expression vector. The full-length and truncated proteins were expressed in Escherichia coli, producing active enzymes of 0.25 and 305 milliunits, respectively, per milliliter of the medium in the lysate of E. coli. The truncated enzyme was much more soluble without detergent than the full-length enzyme. The enzyme catalyzed the transfer of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid to disaccharides, such as lactose and N-acetyllactosamine, with low apparent K(m) and to monosaccharides, such as alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside, with much lower apparent K(m). Thus, this sialyltransferase is unique and should be very useful for achieving high productivity in E. coli with a wide substrate range.
Tsukamoto
Hiroshi
H
Glycotechnology Business Unit, Japan Tobacco Incorporated, Higashibara, Iwata, Shizuoka 438-0802, Japan. hiroshi.tsukamoto@ims.jti.co.jp
Takakura
Yoshimitsu
Y
Yamamoto
Takeshi
T
D000818:Animals; D003001:Cloning, Molecular; D049832:Decapodiformes; D004187:Disaccharides / Q000737:chemistry; D005690:Galactose / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D010776:Photobacterium / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D012794:Sialic Acids / Q000737:chemistry; D012799:Sialyltransferases / Q000737:chemistry* / Q000502:physiology; D013379:Substrate Specificity; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D004187:Disaccharides; D012794:Sialic Acids; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D005690:Galactose; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase