Crystal Structure of human pyridoxal kinase: structural basis of M(+) and M(2+) activation

Abstract

Pyridoxal kinase catalyzes the transfer of a phosphate group from ATP to the 5' alcohol of pyridoxine, pyridoxamine, and pyridoxal. In this work, kinetic studies were conducted to examine monovalent cation dependence of human pyridoxal kinase kinetic parameters. The results show that hPLK affinity for ATP and PL is increased manyfold in the presence of K(+) when compared to Na(+); however, the maximal activity of the Na(+) form of the enzyme is more than double the activity in the presence of K(+). Other monovalent cations, Li(+), Cs(+), and Rb(+) do not show significant activity. We have determined the crystal structure of hPLK in the unliganded form, and in complex with MgATP to 2.0 and 2.2 A resolution, respectively. Overall, the two structures show similar open conformation, and likely represent the catalytically idle state. The crystal structure of the MgATP complex also reveals Mg(2+) and Na(+) acting in tandem to anchor the ATP at the active site. Interestingly, the active site of hPLK acts as a sink to bind several molecules of MPD. The features of monovalent and divalent metal cation binding, active site structure, and vitamin B6 specificity are discussed in terms of the kinetic and structural studies, and are compared with those of the sheep and Escherichia coli enzymes.

Figure 1.

Activity of hPLK in the presence of either Na⁺ or K⁺. Monovalent cations were added in increasing concentration to hPLK in buffered triethanolamine BES, pH 7.3 (closed circles for Na⁺ and triangles for K⁺). Initial velocity was determined at 388 nm, where the product PLP has it maximal absorbance.

Figure 2.

Overall structure of hPLK. (A) The monomeric structure with bound ATP (stick), MPD (stick), Na⁺ (brown sphere), and Mg²⁺ (magenta sphere) at the active site. α-Helices and β-strands are colored yellow and red, respectively. The secondary structures are labeled. (B) The dimeric structure, also with bound ATP (cyan CPK), MPD (gray CPK), Na⁺ (brown sphere), and Mg²⁺ (magenta sphere). Monomers A and B are colored red and yellow, respectively.

Figure 3.

Structural basis of monovalent and divalent ions' activation of hPLK. (A) Na⁺ (brown sphere) bound to the active site of monomer A of unliganded hPLK. The coordination sphere of Na⁺ involves only water molecules (red spheres); with the latter mediating intricate hydrogen-bond interactions with the active site protein residues (sticks). (B) Stereoview of the superimposed active sites of unliganded hPLK (colored yellow) and hPLK-MgATP complex (colored cyan). Three MPD molecules occupy the active site of the unliganded hPLK structure, whereas two are found in the hPLK–MgATP complex. Relevant protein side chains, MPD, and ATP are shown in sticks, and the metal ions are shown in spheres. (C) Na⁺ (brown sphere) and Mg²⁺ (magenta sphere) binding modes at the active site of hPLK–MgATP complex. The protein backbone is shown in blue ribbons, and relevant protein residues are shown in blue sticks. Both metal ions make contact with the protein, ATP (sticks), water molecules (red spheres), and/or MPD molecule (brown stick).

Figure 4.

The active site and substrate binding mode of hPLK–MgATP complex. (A) Schematic diagram showing interactions (dotted lines) between Na⁺, Mg²⁺, ATP, and the protein residues of hPLK. Bond distances are average of the two active sites. (B) Stereoview of the superimposed active sites of hPLK–MgATP (colored yellow) and shPLK–MgATP (colored red). ATP, PM, and MPD molecules, and relevant protein side chains are shown in sticks. Monovalent ions (Na⁺ in hPLK and K⁺ in shPLK) and divalent ions (Mg²⁺ in hPLK and Zn²⁺ in shPLK) are shown in spheres. Note that in the hPLK–MgATP structure, MPD is observed bound at the vitamin B6 site, which overlaps the bound PM in the shPLK–MgATP structure. (C) A Fo−Fc map (contoured at the 3.0 σ level) of Na⁺, Mg²⁺, and ATP of the hPLK–MgATP complex, calculated before the metal ions and ATP were added to the refined model. (D) A 2Fo−Fc map (contoured at the 1.0 σ level) of Na⁺, Mg²⁺, and ATP of the hPLK–MgATP complex. Both maps are superimposed with the final refined models.