In this study, we explored three-dimensional modeling of the human GNE/MNK enzyme and created a model of both GNE-epimerase and MNK-kinase enzymatic domains, as well as a prediction of the putative active sites of these enzymes. Most of the active site residues are conserved and could be assigned functions similar to bacterial homologs. Residues located at the secondary structure interfaces showed a higher degree of homology (42%) compared to overall homology (18–27%) and indicate similar folds.
We identified GNE-domain residues R19, A20, D21, K24, P27, M29, H45, G111, D112, R113, H132, E134, G136, D143, D144, R147, G182, D187, H220, S301, G302, and E307 as putative active site residues. Similarly, we identified residues D413, G416, R420, N516, D517, G545, E566, H569, C579, C581, C586, and E588, as putative MNK active site residues (Table III).
As expected, very low similarity in amino acid composition is observed at the site of bacterial allosteric regulation (which is absent from the mammalian enzyme), at the site of mammalian allosteric regulation (which is absent from the bacterial enzymes), and at the subunit interface.
The active site of the 2-epimerase (GNE) domain is located between domains I (N-terminal) and II (C-terminal), each exhibiting topology of Rossmann dinucleotide binding fold. Most of the secondary structure elements of domains I and II can be aligned (Table II) indicating similarity in their three-dimensional structure.
Differences in the active site residues between enzymes contribute to enzyme specificity toward substrates. For example, S. tokodaii
hexokinase (pdb code 2e2o) phosphorylates glucose, mannose, glucosamine, and GlcNAc while H. sapiens
hexokinase I (pdb code 1dgk) phosphorylates glucose, but does not accommodate GlcNAc. Arthrobacter sp
. glucomannokinase (pdb code 1woq) phosphorylates glucose and mannose. However, not only active site residues but also residues distant from the active site can contribute to specificity. Four of the five residues involved in glucose binding (D195, E244, H247, and E266) are identical in Mlc, Arthrobacter sp
. glucomannokinase, E. coli
glucokinase, H. sapiens
MNK, and ROK family member B. subtilis
fructokinase. The fifth residue, H194 in Mlc, is an asparagine in Arthrobacter sp
. glucomannokinase, H. sapiens
MNK, and E. coli
glucokinase, and a threonine in B. subtilis
fructokinase. However, despite the high similarity of the binding site, Mlc does not bind glucose nor glucose 6-phosphate. Mutation of the histidine to asparagine does not result in glucose binding or glucokinase activity (Schiefner et al. 2005
). Residues at helix–helix interfaces, which position active site residues, may be candidates for this role.
The proposed three-dimensional model of the active site of the kinase domain of the GNE/MNK enzyme assigns a structural role to all catalytic residues due to a high degree of similarity between hexokinases, ROK family kinases, and glucomannokinases.
Further modeling has to address possible orientations of the GNE domain relative to the MNK domain in the bifunctional GNE/MNK enzyme, possible arrangement of its subunits in the active hexameric state, composition of secondary structure interfaces with low homology, structure of the allosteric site, and exact location of ligands in the active sites and allosteric site.
Mutations associated with HIBM and sialuria were mapped onto the preliminary three-dimensional model of the GNE/MNK enzyme. Location of the mutations relative to active sites and secondary structure interfaces assists in predicting effects of these mutations on the enzymatic activity. Practically all mutations either directly interfere with the location of residues important for catalysis or affect secondary structure interfaces and indirectly contribute to the positioning of catalytic residues and binding sites of substrates.
Mutations associated with HIBM and sialuria have proximal (in the active sites and their vicinity) and distal (at the secondary structure interfaces) effects on the structure and function of the enzyme. Mutant p.M712T, being at the interface of α-helices α4 (catalytic) and α10, most likely affects GlcNAc, Mg2+
, and/or ATP binding. Changes in secondary structure content were also observed for this mutant measured by CD spectroscopy (Penner et al. 2006
Modeling of the GNE/MNK, the key enzyme in sialic acid biosynthesis, and related proteins contributes to further understanding of GNE/MNK function, its ligands, and the origin of substrate/inhibitor/regulator specificity of this family of enzymes. Furthermore, modeling of the mutations associated with HIBM or sialuria reveals why these mutations contribute to decreased/inhibited enzymatic activities. It is possible that further modeling studies reveal ligands (with a similar or different structure to ManNAc) that may be good candidates for rescuing the (misfolding) effects of mutated GNE/MNK, or suggest new possible metabolic pathways that may overcome these effects and may be applied for the treatment of HIBM or sialuria.
This work contributes to further understanding of GNE/MNK ligand binding and function, which may assist future studies for therapeutic options that target misfolded GNE/MNK in HIBM and/or sialuria.