A BIFUNCTIONAL ENZYME CATALYZES THE FIRST 2 STEPS IN N-ACETYLNEURAMINIC ACID BIOSYNTHESIS OF RAT-LIVER - MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF UDP-N-ACETYL-GLUCOSAMINE 2-EPIMERASE N-ACETYLMANNOSAMINE KINASE/

N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a g roup of important molecules in biological recognition systems, Biosynt hesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-ac etylglucosamine a-epimerase (UDP-GlcNAc a-epimerase, EC 5.1.3.14)/N-ac etylmannosamine kinase (ManNAc kinase, EC 2.7.1.60) in rat liver (Hind erlich, S., Stasche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chen. 272, 24313-24318), In the present paper we report the isolation and characterization of a cDNA clone encoding this bifunctional enzyme . An open reading frame of 2166 base pairs encodes 722 amino acids wit h a predicted molecular mass of 79 kDa. The deduced amino acid sequenc e contains exact matches of the sequences of five peptides derived fro m tryptic cleavage of the enzyme, The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both epimerase and kin ase activity. Distribution of UDP-GlcNAc 2-epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining bot h specific enzyme activities. Secreting organs (liver, salivary glands , and intestinal mucosa) showed high specific activities of UDP-GlcNAc 2-epimerase/ManNAc kinase, whereas significant levels of these activi ties were absent from other organs (lung, kidney, spleen, brain, heart , skeletal muscle, and testis). Northern blot analysis revealed no UDP -GlcNAc 2-epimerase/ManNAc kinase mRNA in the non-secreting tissues.