Characterization of a recombinant Neisseria meningitidis alpha-2,3-sialyltransferase and its acceptor specificity.
The structure and specificity of the recombinant alpha-2,3-sialyltransferase from Neisseria meninigitidis are reported. This enzyme showed an unusual acceptor specificity in that it could use alpha-terminal and beta-terminal Gal residues as acceptors. In addition (beta1-->4)-linked and (beta1-->3)-linked terminal Gal served as acceptors. These properties distinguish the bacterial enzyme from the more widely investigated mammalian equivalents. The protein was expressed as a membrane-associated protein in Escherichia coli at a level of 750 U/l (approximately 250 mg/l). The protein could be extracted with buffers containing 0.2% Triton X-100 and purified to homogeneity using immobilized-metal-affinity chromatography. Electrospray-ionization mass spectrometry of peptides obtained by cleavage with cyanogen bromide and trypsin confirmed over 95% of the deduced amino acid sequence. When used for enzymatic synthesis in coupled reactions with recombinant CMP-Neu5Ac synthetase, the alpha-2,3-sialyltransferase could sialylate fluorescent derivatives of N-acetyllactosamine with N-acetylneuraminic acid, N-propionylneuraminic acid and N-glycoloylneuraminic acid.
Gilbert
M
M
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Cunningham
A M
AM
Watson
D C
DC
Martin
A
A
Richards
J C
JC
Wakarchuk
W W
WW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D017931:DNA Primers / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000201:enzymology* / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012799:Sialyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D017931:DNA Primers; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase