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J Biol Chem, Vol. 274, Issue 40, 28771-28778, October 1, 1999
From the Institut für Molekularbiologie und Biochemie, Freie
Universität Berlin, Arnimallee 22, D-14195 Berlin-Dahlem, Germany
N-Acetylneuraminic acid is the most
common naturally occurring sialic acid, as well as being the
biosynthetic precursor of this group of compounds. UDP-GlcNAc
2-epimerase/N-acetylmannosamine kinase has been shown to be
the key enzyme of N-acetylneuraminic acid biosynthesis in
rat liver, and it is a regulator of cell surface sialylation. The
N-terminal region of this bifunctional enzyme displays sequence
similarities with prokaryotic UDP-GlcNAc 2-epimerases, whereas the
sequence of its C-terminal region is similar to sequences of members of
the sugar kinase superfamily. High level overexpression of active
enzyme was established by using the baculovirus/Sf9 system. For
functional characterization, site-directed mutagenesis was performed on
different conserved amino acid residues. The histidine mutants H45A,
H110A, H132A, H155A, and H157A showed a drastic loss of epimerase
activity with almost unchanged kinase activity. Conversely, the mutants
D413N, D413K, and R420M in the putative kinase active site lost their kinase activity but retained their epimerase activity. To estimate the structural perturbation effect due to site-directed mutagenesis, the oligomeric state of all mutants was determined by gel filtration analysis. The mutants D413N, D413K, and R420M as well as H45A were
shown to form a hexamer like the wild-type enzyme, indicating little
influence of mutation on protein folding. Histidine mutants H155A and
H157A formed mainly trimeric enzyme with small amounts of hexamer.
Oligomerization of mutants H110A and H132A was also significantly
different from that of the wild-type enzyme. Therefore the loss of
epimerase activity in mutants H110A, H132A, H155A, and H157A can
largely be attributed to incorrect protein folding. In contrast,
the mutation site of mutant H45A seems to be involved directly in
the epimerization process, and the amino acids Asp-413 and Arg-420 of
UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase are
essential for the phosphorylation process. The fact that either epimerase or kinase activity are lost selectively provides evidence for
the existence of two active sites working quite independently.
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