The Biosynthesis of Cytidine 5'-Monophospho-N-acetylneuraminic Acid by an Enzyme from Neisseria meningitidis

In the accompanying paper (2) it was shown that Neisseria meningitidis, strain 1908 (Group C), forms N-acetylneuraminic acid by the condensation of N-acetyl-n-mannosamine and phosphoenolpyruvate with elimination of the phosphate group.In a preliminary communication we have reported a neK type of reaction carried out by another enzyme of Ncisserio meningitidis, strain 1998, which might be considered the activation of N-acetylneuraminic acid (1).In this irreversible reaction, cytidine triphosphate condenses with N-acetylneuraminic acid to form cytidine 5'-monophospho-N-acetylneuraminic acid (Diagram 1)' and inorganic pyrophoaphate (Equation 1). CTP + NAN + CMP-NAN + PP (1)The compound itself, cytidine 5'-monophospho-N-acetylneuraminic acid, was first isolated from Escherichiu coli, K235 (4), a microorganism which also forms colominic acid, a polymer of N-acetylneuraminic acid (5).Recently, Roseman (6) has described the formation of cytidine 5'-monophospho-N-acetylneuraminic acid from N-acetylneuraminic acid and cytidine triphosphate, according to Equation 1, in a mammalian system.This paper reports in detail the isolation, purification, and properties of the enzyme involved and the identification of the products of the reaction. EXPERIMENTAL PROCEDURE Ml&l-i&Sucleotides were purchased from Pabst Laboratories and the California Corporation for Biochemical Research.Sodium borohydride was obtained from Metal Hydrides, Inc., Beverly, Massachusetts.Crystalline N-acetylneuraminic acid was prepared from human plasma proteins (7).Cl'-N-acetylneuraminic acid labeled in the N-acetyl group was synthesized from CWNacetyl-n-mannosamine in a system containing ATP, phosphoenolpyruvate, and rat liver N-acetyl-n-mannosamine kinase and condensing enzyme (8).C14-N-acetyl-n-mannosamine was prepared by the acetylation of n-mannosamine with Cl'-labeled acetic anhydride (9).N-Glycolyneuraminic acid was isolated from trout eggs (10).Equine diacetyl-and bovine triacetylneuraminic acids were the gifts of Professor G. Blis (11).* A preliminary report of this work was given at the Conference on Mucous Secretions sponsored by the New York Academy of Sciences, May 2 to 5, 1962, and in (1).* R. Kuhn and R. Brossmer (3) have recently shown that the 4-hydroxyl