Selective loss of either the epimerase or kinase activity of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase due to site-directed mutagenesis based on sequence alignments

N-Acetylneuraminic acid is the most common naturally occurring sialic acid, as well as being the biosynthetic precursor of this group of compounds. UDP-Glc-NAc 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-Glc-NAc 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 wildtype 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 wildtype 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.