CMP-Sialate Synthetase fromNeisseria meningitidis− Overexpression and Application to the Synthesis of Oligosaccharides Containing Modified Sialic Acids

Abstract

The gene siaB encoding the CMP-sialate synthetase [EC 2.7.7.43] from Neisseria meningitidis serogroup B was subcloned for overexpression in Escherichia coli K12 using the expression vector pKK223–3. With the recombinant strain, 7500 U of synthetase could be produced per liter of cell culture on a 10 L-scale (1350 U/g cells; 20 U/mg protein). Purified enzyme was obtained in high yields (>85%) and with a high specific activity (⩾134 U/mg protein) by an efficient, two-step scheme consisting of DEAE anion-exchange chromatography and ammonium sulfate precipitation. In contrast to known bacterial CMP-sialate synthetases, this enzyme was found to exhibit a broad substrate tolerance, particularly by accepting C5-modified Neu5Ac derivatives as substrates. This included neuraminic acid N-carbamoylated with typical protective groups of different length and bulkiness, an unsaturated acrylamide derivative and the corresponding saturated moiety, as well as the deaminated KDN analogue. Also, the latter structure can be varied by deoxygenation, epimerization at C5 or at the terminal chain, and by shortening the chain length to an octulosonic acid. The high expressivity of the recombinant production clone, the high catalytic efficiency of the enzyme, and its broad substrate tolerance make this synthetase from N. meningitidis the preferred catalyst for the enzymatic synthesis of CMP-Neu5Ac and of derivatives modified in the sialic acid moiety. Several CMP-conjugates made available by this procedure could be transferred effectively onto the acceptor N-acetyllactosamine using the α-2,6-sialyltransferase from rat liver to generate the corresponding trisaccharides.