Molecular modeling of the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase and predictions of structural effects of mutations associated with HIBM and sialuria

Abstract

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ ManNAc kinase (GNE/MNK), encoded by the GNE gene, catalyzes the first two committed, rate-limiting steps in the biosynthesis of N-acetylneuraminic acid (sialic acid). GNE/MNK is feedback inhibited by binding of the downstream product, CMP-sialic acid in its allosteric site. GNE mutations can result in two human disorders, hereditary inclusion body myopathy (HIBM) or sialuria. So far, no active site geometry predictions or conformational transitions involved with function are available for mammalian GNE/MNK. The N-terminal GNE domain is homologous to various prokaryotic 2-epimerases, some of which have solved crystallographic structures. The C-terminal MNK domain belongs to the sugar kinases superfamily; its crystallographic structure is solved at 2.84 A and three-dimensional structures have also been reported for several other kinases. In this work, we employed available structural data of GNE/MNK homologs to model the active sites of human GNE/MNK and identify critical amino acid residues responsible for interactions with substrates. In addition, we modeled effects of GNE/MNK missense mutations associated with HIBM or sialuria on helix arrangement, substrate binding, and enzyme action. We found that all reported mutations are associated with the active sites or secondary structure interfaces of GNE/MNK. The Persian-Jewish HIBM founder mutation p.M712T is located at the interface alpha4alpha10 and likely affects GlcNAc, Mg2+, and ATP binding. This work contributes to further understanding of GNE/MNK function and ligand binding, which may assist future studies for therapeutic options that target misfolded GNE/MNK in HIBM and/or sialuria.

GNE myopathy in India

“…From the predicted structure models reported by Kurochkina, et al, p.Arg420X can preserve its epimerase activity and the enzyme hexameric state, whereas p.Try513X causes its conformational change on glucose binding if these proteins are produced. [23] This study confirms that GNE myopathy may be indeed one among the common inherited myopathies among Indians. Further analysis in a larger cohort is required to clarify the genotype-phenotype correlations of GNE myopathy among Indian population.…”

UDP-GlcNAc 2-Epimerase/ManNAc Kinase (GNE): A Master Regulator of Sialic Acid Synthesis

“…Due to the lack of a crystal structure two different alternative approaches were made. First, models of the structure of the two different functional domains were generated basing on similarities to related enzymes, mostly of prokaryote origin, and their known crystal structures [42, 43]. These studies were motivated in particular by the huge set of mutations causing HIBM and its unclear influence on GNE function (see below).…”

Aberrant O‐GlcNAcylation disrupts GNE enzyme activity in GNE myopathy

“…Removal of O-GlcNAc increases the activity by 25% in the M743T variant, whereas kinase activity of the wild-type GNE is unaffected, indicating that the O-GlcNAc modification of the M743T variant interferes with noncovalent interactions, which are responsible for kinase activity. This theory can be supported by the findings of Tong et al and Kurochkina et al suggesting methionine is involved in substratebinding site of ATP [27,28].…”

“…The reduced activity of the M743T variant supposes an involvement of methionine in the active center or allosteric center of GNE epimerase domain. According to this, Kurochkina et al [27] reported in 2010 that methionine 743 is located at the interface of the a-helices a4 and a10 and likely affects GlcNAc, Mg 2+ , and ATP binding. Tong et al [28] 4.…”