Chemical, genetic and structural assessment of pyridoxal kinase as a drug target in the African trypanosome

Abstract

Pyridoxal-5'-phosphate (vitamin B(6) ) is an essential cofactor for many important enzymatic reactions such as transamination and decarboxylation. African trypanosomes are unable to synthesise vitamin B(6) de novo and rely on uptake of B(6) vitamers such as pyridoxal and pyridoxamine from their hosts, which are subsequently phosphorylated by pyridoxal kinase (PdxK). A conditional null mutant of PdxK was generated in Trypanosoma brucei bloodstream forms showing that this enzyme is essential for growth of the parasite in vitro and for infectivity in mice. Activity of recombinant T. brucei PdxK was comparable to previously published work having a specific activity of 327 ± 13 mU mg(-1) and a K(m)(app) with respect to pyridoxal of 29.6 ± 3.9 µM. A coupled assay was developed demonstrating that the enzyme has equivalent catalytic efficiency with pyridoxal, pyridoxamine and pyridoxine, and that ginkgotoxin is an effective pseudo substrate. A high resolution structure of PdxK in complex with ATP revealed important structural differences with the human enzyme. These findings suggest that pyridoxal kinase is an essential and druggable target that could lead to much needed alternative treatments for this devastating disease.

Fig. 1

Growth characteristics and biochemical analysis of cDKO cells in vitro. A. The growth of the cDKO cell line in PDM was assessed in the presence (open circles) and absence of tetracycline (closed circles). B. Expression of PdxK in cDKO cells was assessed by immunoblotting at various points in the time-course. Trypanosomes (5 × 10⁶ per lane) were probed with antiserum to T. brucei PdxK and to T. brucei TryX as a control.

Fig. 2

Virulence of WT and cDKO T. brucei infections in mice. Results are presented as a Kaplan–Meier survival plot. Symbols: WT cells (×) and cDKO with (•) or without (○) doxycycline in drinking water.

Fig. 3

Recombinant expression of T. brucei PdxK. TbPdxK was expressed from pET3a in BL21 (DE3) cells. A. SDS-PAGE analysis, 10 µg protein was loaded per lane. Lane 1, clarified lysate; lane 2, fractions pooled from the Q-sepharose column; lane 3, fractions pooled from gel filtration. B. Gel filtration of Q-sepharose-purified TbPdxK. The inset shows a plot of elution volume vs. log molecular mass of a mixture of protein standards. The closed circle represents the elution volume of TbPdxK.

Fig. 4

Assay optimization. PdxK activity was measured by increase in absorbance at 388 nm resulting from formation of PLP in 100 mM HEPES buffer, pH 7.4 and 1.5 mM ATP. A. Divalent cations were tested at 100 µM in the presence of 300 µM pyridoxal in duplicate. B. The two cations conferring the highest activity (MgCl₂ and MnCl₂) were titrated from 1 mM to 5 µM in duplicate. C. Kinetic behaviour of the enzyme was determined in the presence of 250 µM MgCl₂ (closed circles) or 150 µM ZnCl₂ (open squares). Data were fitted to the equation describing high substrate inhibition as described in the experimental procedures.

Fig. 5

Effect of ginkgotoxin on T. brucei cells. A. An example of an EC₅₀ determination is shown. Microplates were seeded with 1 × 10³ cells ml⁻¹ and incubated in the presence of Ginkgotoxin (50 µM to 23 nM) for 3 days. Cell proliferation was assayed with resazurin. EC₅₀ values were determined on three separate occasions. B. Mean EC₅₀ values, weighted to the standard error, for the parental ‘single marker’ line (WT) are compared with those for the single-allele PdxK knockout (SKO) and the conditional null mutant.

Fig. 6

Structure of T. brucei PdxK in complex with ATP. A. TbPdxK monomeric subunit with secondary structure annotated according to Fig. 7. α-Helices are in orange, β-strands are in magenta, the 3₁₀-helix is in green and ATP is shown with carbon atoms in green, nitrogen atoms in blue, oxygen atoms in red, and phosphorus atoms in yellow. Figure produced using PyMOL (DeLano, 2005). B. The dimeric assembly of TbPdxK annotated as in panel A.

Fig. 7

Sequence alignment of TbPdxK with human PdxK. Conserved residues are highlighted in black. Sequences are numbered according to TbPdxK. The secondary structure of TbPdxK is indicated on the top line with α-helices in orange, β-strands in magenta, and 3₁₀-helices in green. Figure produced using ALINE (Bond and Schuttelkopf, 2009).

Fig. 8

Important residues in and around the substrate and cofactor binding sites of parasite and human PdxK. A. T. brucei PdxK. B. Human PdxK (Gandhi et al., 2009). Oxygen atoms are red, nitrogen atoms blue, phosphorus atoms orange, and magnesium grey. Hydrogen bonds are shown as black dotted lines. Important residues, ATP, and pyridoxal-5′-phosphate (PLP) are labelled.