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

Chia Shin Yang,Tzu-Ping Ko,Pin-Kuei Fu,Ching-Heng Lin,Shu-Jung Lai,Tzu-Hung Hsiao,Chi-Hung Huang,Yeh Chen,Sheng-Chia Chen

doi:10.1038/srep23274

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.

Highlights

The sialic acids are a family of nine-carbon sugar derived from neuraminic acid[1] (Fig. 1a)
Being a key enzyme that catalyzes the rate-limiting step of sialic acid biosynthesis, GlcNAc 2-epimerase/ManNAc kinase (GNE) plays an important role in regulation of cell-surface sialyation level by binding to the downstream product CMP-Neu5Ac
The non-hydrolyzing enzyme participates in teichoic acid biosynthesis, and it is positively regulated by UDP-GlcNAc20

Summary

Introduction

The sialic acids are a family of nine-carbon sugar derived from neuraminic acid[1] (Fig. 1a). Sialic acid biosynthesis in mammals starts by converting UDP-GluNAc into UDP and ManNAc, followed by phosphorylation of ManNAc at the sixth position (Fig. 1b). Catalysis of both reactions is carried out by the bifunctional enzyme GNE, which in human is composed of ~720 amino acid residues and divided into the epimerase part (~400 aa) and the kinase part (~300 aa)[7]. 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. The feedback inhibition is highly positively cooperative and it does not affect the ManNAc kinase activity[9]. The binding of UDP-GlcNAc to an allosteric site triggered domain closure and activates the enzyme by forming a proper substrate-binding pocket

Methods

Results

Conclusion