PDBsum entry 2a2c

Transferase

PDB id: 2a2c

Contents

Protein chain

446 a.a.

Ligands

NG1

ADP

Metals

_MG

_CL

_NA

Waters ×457

References listed in PDB file

Key reference

Title

The molecular architecture of human n-Acetylgalactosamine kinase.

Authors

J.B.Thoden, H.M.Holden.

Ref.

J Biol Chem, 2005, 280, 32784-32791. [DOI no: 10.1074/jbc.M505730200]

PubMed id

16006554

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.

Figure 1.
FIGURE 1. Ribbon representation of human GalNAc kinase. Human GalNAc kinase folds into two motifs referred to as the N- and C-terminal domains. As indicated in a, the N-terminal domain is dominated by a seven-stranded mixed -sheet highlighted in magenta. Two layers of antiparallel -sheet, colored in yellow and blue, characterize the C-terminal domain. The location of the active site is indicated by the ball-and-stick representations for GalNAc-1-phosphate and MgADP, with the Mg2+ ion highlighted in green. Electron density corresponding to GalNAc 1-phosphate and MgADP is displayed in b. While the protein was crystallized in the presence of GalNAc and MgATP, the electron density clearly reveals that the enzyme is active in the crystalline lattice. The map, contoured at 2 , was calculated with coefficients of the form F[o] - F[c], where F[o] was the native structure factor amplitude, and F[c] was the calculated structure factor amplitude from the model lacking coordinates for the ligands. A close-up view of the active site with bound products is depicted in c. Only those residues located within 3.2 Å of the ligands are shown. The green dashed lines indicate coordinate covalent bonds between the magnesium ion and its ligands. The black dashed lines indicate potential hydrogen bonding interactions. For clarity, Ser141 and Gly143 were omitted from the figure.

Figure 3.
FIGURE 3. A comparison of human galactokinase versus GalNAc kinase. The human forms of galactokinase and GalNAc kinase differ in three specific regions labeled A, B, and C and indicated by the blue ribbons in a. A superposition of the sugar binding regions for these enzymes is given in b. The yellow bonds correspond to GalNAc kinase, with the sugar ligand depicted in aquamarine. The white bonds correspond to galactokinase, with the sugar ligand highlighted in magenta. The red and black labels correspond to residues in galactokinase and GalNAc kinase, respectively. Coordinates for the human galactokinase were from this laboratory (Protein Data Bank accession number 1WUU [PDB] ).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 32784-32791) copyright 2005.