The Molecular Architecture of Human N-Acetylgalactosamine Kinase

James B Thoden,Hazel M Holden

doi:10.1074/jbc.m505730200

Abstract

Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.

Highlights

The galactokinases fold into two distinct N- and C-terminal domains with their active sites wedged between these motifs
A second gene encoding a putative galactokinase, with ϳ35% amino acid sequence identity, was subsequently reported that mapped to chromosome 15, suggesting, perhaps, that humans contain two galactokinases [12]. These apparently contradicting reports for the chromosomal location of human galactokinase have been reconciled through the recent studies of Pastuszak et al [13, 14], who demonstrated that the gene located on chromosome 15 does not, encode a bona fide galactokinase but rather a GalNAc kinase (Scheme 1)
In an effort to understand the observed substrate specificity of the enzyme, we initiated a structural analysis of human GalNAc kinase

Summary

Structure of GalNAc Kinase

SCHEME I purchased from ATCC such that the forward and reverse primers added NdeI and XhoI cloning sites, respectively. The PCR product was purified with the QIAquick PCR purification kit (Qiagen), followed by A-tailing and ligation into pGEM-T vector (Promega) This vector was used to transform Escherichia coli DH5␣ cells. Crystals were obtained in the presence of GalNAc and MgATP (which turned over to product in the active site) Whereas these belonged to the same space group, P65, the unit cell dimensions were slightly different at a ϭ b ϭ 124 Å, and c ϭ 60 Å. Cultures were grown at 37 °C to an optical density of ϳ0.9 at 600 nm and cooled for 10 min in an ice water bath They were transferred to an incubator at 16 °C, and each flask was supplemented with 50 mg each of lysine, threonine, and phenylalanine and 25 mg each of leucine, isoleucine, valine, and selenomethionine.

Independent reflections

RESULTS AND DISCUSSION

Weighted root mean square deviations from ideality