Characterization of a Recombinant Neisseria Meningitidesα‐2,3‐Sialyltransferase and its Acceptor Specificity

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Abstract
The structure and specificity of the recombinant α‐2,3‐sialyltransferase from Neisseria meningitides are reported. This enzyme showed an unusual acceptor specificity in that it could use α‐terminal and β‐terminal Gal residues as acceptors. In addition (β→4)‐linked and (β→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 (≈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 α‐2,3‐sialyltransferase could sialylate fluorescent derivatives of N‐acetyllactosamine with N‐acetylneuraminic acid, N‐propionylneuraminic acid and N‐glycoloylneuraminic acid.