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 meningitidis are reported. This enzyme showed an unusual acceptor specificity in that it could use alpha-terminal and beta-terminal Gal residues as accepters. In addition (beta 1-->4)-linked and (beta 1-->3)-linked terminal Gal served as accepters. 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 (approximate to 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.