CMP-sialate synthetase from Neisseria meningitidis - Overexpression and application to the synthesis of oligosaccharides containing modified sialic acids
M. Knorst et Wd. Fessner, CMP-sialate synthetase from Neisseria meningitidis - Overexpression and application to the synthesis of oligosaccharides containing modified sialic acids, ADV SYNTH C, 343(6-7), 2001, pp. 698-710
The gene siaB encoding the CMP-sialate synthetase [EC 2.7.7.43] from Neisse
ria meningitidis serogroup B was subcloned for overexpression in Escherichi
a coli K12 using the expression vector pKK223-3. With the recombinant strai
n, 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 (greater than or equ
al to 134 U/mg protein) by an efficient, two-step scheme consisting of DEAE
anion-exchange chromatography and ammonium sulfate precipitation. In contr
ast to known bacterial CMP-sialate synthetases, this enzyme was found to ex
hibit a broad substrate tolerance, particularly by accepting C5-modified Ne
u5Ac derivatives as substrates. This included neuraminic acid N-carbamoylat
ed with typical protective groups of different length and bulkiness, an uns
aturated acrylamide derivative and the corresponding saturated moiety, as w
ell as the deaminated KDN analogue. Also, the latter structure call be vari
ed by deoxygenation, epimerization at C5 or at the terminal chain, and by s
hortening the chain length to an octulosonic acid. The high expressivity of
the recombinant production clone, the high catalytic efficiency or the enz
yme, and its broad substrate tolerance make this synthetase from A meningit
idis the preferred catalyst for the enzymatic synthesis of CMP-Neu5Ac and o
f derivatives modified in the sialic acid moiety. Several CMP-conjugates ma
de available by this procedure could be transferred effectively onto the ac
ceptor N-acetyllactosamine using the alpha -2,6-sialyl-transferase from rat
liver to generate the corresponding trisaccharides.