A bifunctional enzyme catalyzes the first two 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 group of important molecules in biological recognition systems. Biosynthesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1. 3.14)/N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60) in rat liver (Hinderlich, S., Stäsche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 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 with a predicted molecular mass of 79 kDa. The deduced amino acid sequence contains exact matches of the sequences of five peptides derived from tryptic cleavage of the enzyme. The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both epimerase and kinase activity. Distribution of UDP-GlcNAc 2-epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining both 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 activities 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.
Stäsche
R
R
Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Arnimallee 22, D-14195 Berlin-Dahlem, Germany.
Hinderlich
S
S
Weise
C
C
Effertz
K
K
Lucka
L
L
Moormann
P
P
Reutter
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C018765:N-acylmannosamine kinase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; C083982:wecB protein, E coli; D019158:N-Acetylneuraminic Acid