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

Abstract

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 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.

Reduction of UDP-N-acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase Activity and Sialylation in Distal Myopathy with Rimmed Vacuoles

“…ManNAc kinase activity was retained in the N-terminal mutants C13S, H132Q, D176V, D177C, V331A, and D378Y, whereas the C-terminal mutants I472T, V572L, A630T, A631V, and G703S showed dramatic reductions in activities. These data were essentially compatible with a prior report (13). Interestingly, the A524V mutant preferentially affected UDP-GlcNAc 2-epimerase activity, although the mutation is in the kinase domain.…”

“…Enzymatic Activities of GNE Mutants in DMRV PatientsSite-directed mutagenesis of the GNE protein has shown that the two enzymatic activities are separately and independently catalyzed by two domains, an N-terminal epimerase domain and a C-terminal kinase domain (13). Previously, we reported reductions in UDP-GlcNAc 2-epimerase activities in leukocytes from the patients with mutations in the GNE gene (8).…”

“…UDP-GlcNAc 2-epimerase activity was measured as described previously. The ManNAc kinase assay was performed with slight modification according the previous report (13).…”

“…Oligomerization of GNE Mutants-GNE protein forms a homohexamer by oligomerization (13). We examined whether the mutations affect the oligomerization of GNE molecules, because homohexamer structure of GNE protein was reported to be essential for UDP-GlcNAc 2-epimerase activity (13).…”

“…3) Why do mutant proteins not complement each other in patients who have heterozygous mutations in each of the two domains? The two domains in GNE protein have been reported to catalyze the enzymatic reactions separately and independently (13). To address these questions, we studied the relationships between mutations and enzymatic activity using in vitro expression and enzymatic assay systems.…”