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. UD P-Glc-NAc 2-epimerase/N-acetylmannosamine kinase has been shown to be the k ey enzyme of N-acetylneuraminic acid biosynthesis in rat liver, and it is a regulator of cell surface sialylation. The N-terminal region of this bifun ctional enzyme displays sequence similarities with prokaryotic UDP-Glc-NAc 2-epimerases, whereas the sequence of its C-terminal region is similar to s equences of members of the sugar kinase superfamily, High level overexpress ion of active enzyme was established by using the baculovirus/Sf9 system. F or 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 a lmost unchanged kinase activity. Conversely, the mutants D413N, D413K, and R420M in the putative kinase active site lost their kinase activity but ret ained their epimerase activity. To estimate the structural perturbation eff ect due to site-directed mutagenesis, the oligomeric state of all mutants w as determined by gel filtration analysis. The mutants D413N, D413K, and R42 0M as well as H45A were shown to form a hexamer like the wildtype enzyme, i ndicating little influence of mutation on protein folding. Histidine mutant s H155A and H157A formed mainly trimeric enzyme with small amounts of hexam er, Oligomerization of mutants H110A and H132A was also significantly diffe rent from that of the wildtype enzyme. Therefore the loss of epimerase acti vity in mutants H110A, H132A, H155A, and H157A can largely be attributed to incorrect protein folding. In contrast, the mutation site of mutant H45A s eems to be involved directly in the epimerization process, and the amino ac ids Asp-413 and Arg-420 of UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinas e are essential for the phosphorylation process. The fact that either epime rase or kinase activity are lost selectively provides evidence for the exis tence of two active sites working quite independently.