Dataset Information

Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis.

ABSTRACT: 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 feed-back inhibited by the downstream product CMP-Neu5Ac. Here 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. By comparing the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, we propose a possible explanation for the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. On the other hand, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria. Moreover, full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme.

SUBMITTER: Chen SC

PROVIDER: S-EPMC4793188 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis.

Chen Sheng-Chia SC Huang Chi-Hung CH Lai Shu-Jung SJ Yang Chia Shin CS Hsiao Tzu-Hung TH Lin Ching-Heng CH Fu Pin-Kuei PK Ko Tzu-Ping TP Chen Yeh Y

Scientific reports 20160316

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 feed-back inhibited by the downstream product CMP-Neu5Ac. Here 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 confor ...[more]

PMID: 26980148

Similar Datasets

Project description:UDP-glucuronic acid (UDP-GlcA) is a central precursor in sugar nucleotide biosynthesis and common substrate for C4-epimerases and decarboxylases releasing UDP-galacturonic acid (UDP-GalA) and UDP-pentose products, respectively. Despite the different reactions catalyzed, the enzymes are believed to share mechanistic analogy rooted in their joint membership to the short-chain dehydrogenase/reductase (SDR) protein superfamily: Oxidation at the substrate C4 by enzyme-bound NAD+ initiates the catalytic pathway. Here, we present mechanistic characterization of the C4-epimerization of UDP-GlcA, which in comparison with the corresponding decarboxylation has been largely unexplored. The UDP-GlcA 4-epimerase from Bacillus cereus functions as a homodimer and contains one NAD+ /subunit (kcat = 0.25 ± 0.01 s-1 ). The epimerization of UDP-GlcA proceeds via hydride transfer from and to the substrate's C4 while retaining the enzyme-bound cofactor in its oxidized form (≥ 97%) at steady state and without trace of decarboxylation. The kcat for UDP-GlcA conversion shows a kinetic isotope effect of 2.0 (±0.1) derived from substrate deuteration at C4. The proposed enzymatic mechanism involves a transient UDP-4-keto-hexose-uronic acid intermediate whose formation is rate-limiting overall, and is governed by a conformational step before hydride abstraction from UDP-GlcA. Precise positioning of the substrate in a kinetically slow binding step may be important for the epimerase to establish stereo-electronic constraints in which decarboxylation of the labile β-keto acid species is prevented effectively. Mutagenesis and pH studies implicate the conserved Tyr149 as the catalytic base for substrate oxidation and show its involvement in the substrate positioning step. Collectively, this study suggests that based on overall mechanistic analogy, stereo-electronic control may be a distinguishing feature of catalysis by SDR-type epimerases and decarboxylases active on UDP-GlcA.

Dataset Information

Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis.

Publications

Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets