Structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae

Abstract

In most organisms, efficient D-galactose utilization requires the highly conserved Leloir pathway that converts D-galactose to D-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of D-galactose assimilation have been identified. In the so-called De Ley-Doudoroff pathway, D-galactose is metabolized into pyruvate and D-glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-oxo-3-deoxygalactonate kinase, with ATP serving as a phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley-Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein.

Figure 1

d-Galactose metabolic pathways.

Figure 2

Overall structure of 2-oxo-3-deoxygalactonate kinase (stereoview). Green and blue colors indicate the conserved βββαβαβα ASKHA core within the N- and C-terminal domains, respectively. The β₄–β₅ and β₅–α₃ insertions in the N-terminal domain are shown in yellow and orange. Cyan elements represent the β₃–α₁ insertion in the C-terminal domain. Secondary-structure elements have been annotated according to DSSP (Kabsch & Sander, 1983 ▶).

Figure 3

Multiple sequence alignment of selected KDGal kinase homologs. Identical residues are highlighted in red and similar residues are shown as red letters. Secondary-structure elements derived from K. pneumoniae kinase are depicted by arrows (representing β-strands) and cylinders (representing helices). The color code is as in Fig. 2 ▶. All β-strands (S) and helices (H for α-helix, G for 3₁₀-helix) are numbered consecutively. Additionally, secondary-structure elements building the common ASKHA-family core are labeled with Greek letters. The positions of ASKHA signature motifs are marked. The sequences belong to Gram-positive bacteria (Kpneu, K. pneumoniae, current structure, UniProt accession code A6TFZ6; Ecoli, Escherichia coli, B5QUP1; Sente, Salmonella enterica, B5QUP1; Crode, Citrobacter rodentium, D2THW7; Retli, Rhizobium etli, Q2KBX1) and Gram-negative bacteria (Pfluo, Pseudomonas fluorescens, UniProt accession code Q3K8P2; Ropac, Rhodococcus opacus, C1AVX8; Msmeg, Mycobacterium smegmatis, A0R5F7; Seryt, Saccharopolyspora erythraea, A4FCZ7).

Figure 4

The putative substrate- and phosphate-binding site of KDGal kinase. Green color represents molecule A and blue color corresponds to molecule B. To indicate general positions for nucleotide and substrate binding, a β-l-fructose molecule (Fru) and a fragment of an ADP molecule from the l-rhamnulose kinase structure (PDB code 2cgj, molecule A) are shown in ball-and-stick representation. Selected secondary-structure elements are marked.

Figure 5

Oligomerization of 2-oxo-3-deoxygalactonate kinase. (a) A–B dimer (left). Molecule A is shown in green and molecule B is shown in blue. For comparison, the functional dimer of pantothenate kinase in complex with ADP and pantothenate (Pan) is presented (right; PDB entry 3bf1; chain A, purple; chain B, pink; Yang et al., 2008 ▶). Ligand molecules are depicted in sphere representation. Selected secondary-structure elements are marked. (b) A–C dimer. Molecule A is shown in green and molecule C is shown in red. (c) KDGal kinase tetramer (molecule A, green; molecule B, blue; molecule C, red; molecule D, yellow).

Figure 6

Comparison of 2-oxo-3-deoxygalactonate kinase with similar structures. (a) Superposition of KDGal kinase (chain A, green) and butyrate kinase 2 in complex with 5′-adenylyl-(β,γ-methylene)diphosphonate (ACP) (PDB entry 1saz; gray; Diao & Hasson, 2009 ▶). The nucleotide ligand is shown in sphere representation. (b). Superposition of KDGal kinase (chain A, green) and pantothenate kinase in complex with ADP and pantothenate (PDB code 3bf1; chain A, purple; Yang et al., 2008 ▶). For clarity, the pantothenate moiety is not shown. The nucleotide ligand is shown in sphere representation.

Figure 7

Putative nucleotide-binding site of KDGal kinase. The protein molecule is represented by electrostatic surface potential, in which the blue color corresponds to positively charged areas, red to negatively charged areas and white to neutral regions. In the absence of natural ligand, the pockets of molecules B (a) and D (b) are occupied by glycerol molecules soaked into the crystal lattice during cryoprotection. The glycerol moieties are shown in ball-and-stick representation in a 2F _o − F _c electron-density map contoured at the 1σ level.