Identification of the sequence encoding N-acetylneuraminate-9-phosphate phosphatase

Glycobiology. 2006 Feb;16(2):165-72. doi: 10.1093/glycob/cwj050. Epub 2005 Oct 19.


The synthesis of N-acetylneuraminate (Neu5Ac), the main form of sialic acid, proceeds in vertebrates through the condensation of N-acetylmannosamine 6-phosphate and phosphoenolpyruvate to Neu5Ac-9-phosphate, followed by the dephosphorylation of the latter by a specific phosphatase. The sequence encoding Neu5Ac-9-phosphate phosphatase (Neu5Ac-9-Pase; E.C. has not been determined until now. In this work, we have purified Neu5Ac-9-Pase more than 1000-fold from rat liver. Its dependency on Mg2+ and the fact that it was inhibited by vanadate and Ca2+ suggested that it belonged to the haloacid dehalogenase family of phosphatases. Trypsin digestion and mass spectrometry analysis of a polypeptide of about 30 kDa that co-eluted with the activity in the last purification step indicated the presence of a protein designated "haloacid dehalogenase-like hydrolase domain containing 4." The human ortholog of this protein is encoded by a 2-exon gene present on chromosome 20p11. The human protein was overexpressed in Escherichia coli as a fusion protein with a polyHis tag and purified to homogeneity. The recombinant enzyme displayed a >230-fold higher catalytic efficiency on Neu5Ac-9-phosphate than on its second best substrate. Its properties were similar to those of the enzyme purified from rat liver. Neu5Ac inhibited the enzymatic activity by 50% at 15 mM, indicating that no significant inhibition is exerted at physiological concentrations of Neu5Ac. The identification of the gene encoding Neu5Ac-9-Pase will facilitate studies aimed at testing its potential implication in unexplained forms of glycosylation deficiency.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Liver / enzymology
  • Molecular Sequence Data
  • Phosphoric Monoester Hydrolases / chemistry*
  • Phosphoric Monoester Hydrolases / isolation & purification
  • Rats
  • Recombinant Proteins / metabolism


  • Enzyme Inhibitors
  • Recombinant Proteins
  • Phosphoric Monoester Hydrolases
  • N-acylneuraminate-9-phosphatase