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Glycobiology, 2002, Vol. 12, No. 2 65-71
© 2002 Oxford University Press

Purification and characterization of N-acetylneuraminic acid-9-phosphate synthase from rat liver

Hao Chen2,3, Astrid Blume2, Martin Zimmermann-Kordmann2, Werner Reutter2 and Stephan Hinderlich1,2

2Institute of Molecular Biology and Biochemistry, Free University of Berlin, Arnimallee 22, 14195 Berlin-Dahlem, Germany, and 3College of Life Science, Nanjing University, Nanjing 210093, People’s Republic of China

Sialic acids are a group of carboxylated amino sugars important for a variety of cellular functions. N-Acetylneuraminic acid (Neu5Ac) is the predominant sialic acid in nature. Neu5Ac-9-phosphate synthase catalyzes the formation of Neu5Ac-9-phosphate from N-acetylmannosamine-6-phosphate and phosphoenolpyruvate. Neu5Ac-9-phosphate synthase was purified 11,700-fold from rat liver cytosol to apparent homogeneity by ammonium sulfate precipitation, chromatography on hydroxylapatite, phenyl-Sepharose, MonoQ, and finally gel filtration. SDS–PAGE and gel filtration chromatography indicated that the enzyme is a dimer composed of 37-kDa subunits. Analysis of trypic peptides by MALDI-TOF MS verified a high sequence similarity to the corresponding murine enzyme. The Km values of Neu5Ac-9-phosphate synthase were 35 µM for N-acetylmannosamine-6-phosphate and 100 µM for phosphoenolpyruvate. The enzyme displayed an absolute requirement for divalent cations, Mn2+, Fe2+, and Mg2+ being the most effective. In contrast to human Neu5Ac-9-phosphate synthase, the rat enzyme did not utilize mannose-6-phosphate in the synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid 9-phosphate. Neu5Ac-9-phosphate synthase was inactivated by the sulfhydryl modifying reagents, 5,5'-dithio-bis (2-nitrobenzoic acid) and N-ethylmaleimide, and protected from inactivation by the presence of the substrate phosphoenolpyruvate, but not by the presence of N-acetylmannosamine-6-phosphate, showing that at least one cysteine residue is located in the active site of the enzyme.

1 To whom correspondence should be addressed


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