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Literature summary for 3.2.1.183 extracted from

  • Chen, S.C.; Huang, C.H.; Lai, S.J.; Yang, C.S.; Hsiao, T.H.; Lin, C.H.; Fu, P.K.; Ko, T.P.; Chen, Y.
    Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis (2016), Sci. Rep., 6, 23274 .
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

Crystallization (Comment) Organism
enzyme in complex with UDP-GlcNAc, X-ray diffraction crystal structure determination and analysis at 2.7 A resolution. Only UDP is observed in the active site of each monomer despite the use of UDP-GlcNAc in crystallization, presumably the substrate has been converted to ManNAc and UDP before it is trapped in the crystal Homo sapiens

Protein Variants

Protein Variants Comment Organism
D112A site-directed mutagenesis, the mutant shows 97.7% reduced activity compared to the wild-type enzyme Homo sapiens
D143A site-directed mutagenesis, inactive mutant Homo sapiens
E134A site-directed mutagenesis, inactive mutant Homo sapiens
R113A site-directed mutagenesis, inactive mutant Homo sapiens
S302A site-directed mutagenesis, the mutant shows 87.1% reduced activity compared to the wild-type enzyme Homo sapiens

Inhibitors

Inhibitors Comment Organism Structure
CMP-Neu5Ac feedback inhibition, cooperative binding mode and binding structure, overview Homo sapiens
UDP the binding mode of UDP reveals unique interactions with the hydrolyzing epimerase, binding structure, overview. The base is sandwiched between the side chains of Arg19 and Phe287, while making two hydrogen bonds to the backbone of Val282. The two hydroxyl groups of ribose are hydrogen bonded to the side chains of Ser23 and Glu307. The diphosphate forms salt bridges to Arg19, Arg113 and Arg321. It also interacts with His220 and Asn253 through hydrogen bonds, as well as the two consecutive Ser301 and Ser302 at the N-terminus of helix alpha12 Homo sapiens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0005
-
UDP-N-acetyl-alpha-D-glucosamine recombinant mutant D112A, pH 7.5, 37°C Homo sapiens
0.0121
-
UDP-N-acetyl-alpha-D-glucosamine recombinant mutant S302A, pH 7.5, 37°C Homo sapiens
0.0331
-
UDP-N-acetyl-alpha-D-glucosamine recombinant wild-type enzyme, pH 7.5, 37°C Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
UDP-N-acetyl-alpha-D-glucosamine + H2O Homo sapiens
-
N-acetyl-D-mannosamine + UDP
-
?

Organism

Organism UniProt Comment Textmining
Homo sapiens Q9Y223
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
UDP-N-acetyl-alpha-D-glucosamine + H2O
-
Homo sapiens N-acetyl-D-mannosamine + UDP
-
?

Subunits

Subunits Comment Organism
dimer
-
Homo sapiens
More the full-length enzyme can form a dimer or tetramer, depending on the presence of UDP-GlcNAc and CMP-Neu5Ac. GNE forms a closed tetramer in the presence of UDP-GlcNAc and CMP-Neu5Ac. The complex crystal structure of the N-terminal epimerase part of human GNE shows a tetramer in which UDP binds to the active site and CMP-Neu5Ac binds to the dimer-dimer interface. The enzyme is locked in a tightly closed conformation. Each dimer further forms a tetramer with a crystallographic dyad-related dimer Homo sapiens
tetramer dimer of dimers Homo sapiens

Synonyms

Synonyms Comment Organism
GNE
-
Homo sapiens
UDP-GlcNAc 2-epimerase
-
Homo sapiens
UDP-GlcNAc 2-epimerase/ManNAc kinase
-
Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.076
-
UDP-N-acetyl-alpha-D-glucosamine recombinant mutant D112A, pH 7.5, 37°C Homo sapiens
0.791
-
UDP-N-acetyl-alpha-D-glucosamine recombinant mutant S302A, pH 7.5, 37°C Homo sapiens
11.8
-
UDP-N-acetyl-alpha-D-glucosamine recombinant wild-type enzyme, pH 7.5, 37°C Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Homo sapiens

General Information

General Information Comment Organism
malfunction defective GNE inhibition by CMP-Neu5Ac causes cytoplasmic accumulation and increased excretion of free sialic acid. Sialuria is an autosomal dominant disorder which is related to GNE mutation in one of the two arginine residues 263 and 266 (R263L, R266Q or R266W), the mutations in Arg263 and Arg266 can cause sialuria by hindering the enzyme inhibition through CMP-Neu5Ac binding Homo sapiens
metabolism sialic acid biosynthesis in mammals starts by converting UDP-GluNAc into UDP and ManNAc, followed by phosphorylation of ManNAc at the sixth position, catalysis of both reactions is carried out by the bifunctional enzyme GNE. Regulation of cell-surface sialyation level by binding to the downstream product CMP-Neu5Ac. The feedback inhibition is highly positively cooperative and it does not affect the ManNAc kinase activity Homo sapiens
additional information the comparison of the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases might explain the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. Full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme, possible epimerase-kinase channel Homo sapiens
physiological function the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) plays a key role in sialic acid production. It is different from the non-hydrolyzing enzymes for bacterial cell wall biosynthesis, and it is feedback inhibited by the downstream product CMP-Neu5Ac. Being a key enzyme that catalyzes the rate-limiting step of sialic acid biosynthesis, GNE plays an important role in regulation of cell-surface sialyation level by binding to the downstream product CMP-Neu5Ac. Regulation of cell-surface sialyation level by binding to the downstream product CMP-Neu5Ac. The feedback inhibition is highly positively cooperative and it does not affect the ManNAc kinase activity. By mediating cell-cell recognition, sialic acids are important in the development of nervous system. Structure and biosynthesis of sialic acid, overview Homo sapiens

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
65.4
-
UDP-N-acetyl-alpha-D-glucosamine recombinant mutant S302A, pH 7.5, 37°C Homo sapiens
152
-
UDP-N-acetyl-alpha-D-glucosamine recombinant mutant D112A, pH 7.5, 37°C Homo sapiens
356.5
-
UDP-N-acetyl-alpha-D-glucosamine recombinant wild-type enzyme, pH 7.5, 37°C Homo sapiens