| 2004 |
PDX1 (PfPdx1) and PfPdx2 together catalyze de novo vitamin B6 biosynthesis in P. falciparum; the two proteins act together in glutaminase activity with a specific activity of 209 nmol min⁻¹ mg⁻¹ and a Km for glutamine of 1.3 mM. Complementation of pyridoxine-auxotrophic yeast confirmed functional activity of PfPdx1 in vivo. |
Recombinant protein biochemistry (glutaminase assay), yeast complementation |
The Journal of biological chemistry |
High |
15590634
|
| 2005 |
PDX1 (PfPdx1) is the pyridoxal-5'-phosphate synthase catalytic domain; it requires complex formation with PfPdx2 (glutaminase subunit) for catalytic activity of either domain. PDX1 can form a chimeric bi-enzyme with bacterial YaaE (a Pdx2 ortholog) both in vivo and in vitro, but this chimera does not attain full catalytic activity, indicating species-specific structural features govern partner interaction. |
Recombinant protein biochemistry, in vitro reconstitution, chimeric protein assay, crystal structure of Pdx2 at 1.62 Å |
The Journal of biological chemistry |
High |
16339145
|
| 2007 |
Complex formation between PDX1 (PfPdx1) and PfPdx2 is governed by hydrophobic interactions at the interface; l-glutamine binding increases the tightness of the PfPdx1–PfPdx2 interaction ~30-fold. The N-terminal αN-helix of PfPdx1 is required for complex formation; a 'swap mutant' in which the αN-helix was replaced with the B. subtilis segment gained cross-binding to BsPdx2 and partially elicited BsPdx2 glutaminase activity, linking complex interface formation to catalytic activation of the glutaminase. |
Isothermal titration calorimetry (ITC), 3D homology modelling, swap mutagenesis with glutaminase activity assay |
Journal of molecular biology |
High |
17950752
|
| 2008 |
Residues R85, H88, and E91 at the PDX1:PDX1 interface are required for dodecameric complex assembly; alanine substitutions at these positions impede both PDX1 activity and PfPdx2 binding, yielding a monomeric protein. Catalytic-site residues D26, K83, and K151 are required for enzymatic activity; mutation to alanine results in a hexameric (not dodecameric) assembly and does not affect Pdx2 binding or its activity. K151 is specifically required for the transition from hexamer to functional dodecamer. |
Site-directed mutagenesis, gel filtration (oligomeric state analysis), enzyme activity assay, co-purification with PfPdx2 |
PloS one |
High |
18350152
|
| 2009 |
Glycine 155 of PDX1 is required for the transition from hexameric to dodecameric assembly; a G155A substitution results in a hexameric protein with altered secondary structure (by CD) that retains the ability to bind PfPdx2 and support its glutaminase activity, but cannot form the active dodecamer, demonstrating that structural flexibility at this position is necessary for dodecamer formation and full enzymatic activity. |
Static and dynamic light scattering, circular dichroism, co-purification, enzyme assay |
Biochimica et biophysica acta |
High |
19272411
|
| 2010 |
The C-terminus of PDX1 has four distinct and separable functions: assembly of PDX1 monomers, binding of the pentose substrate ribose 5-phosphate (R5P), formation of the reaction intermediate I₃₂₀, and final PLP synthesis. PLP formation is the only function conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex. |
C-terminal deletion analysis, biochemical assays for intermediate formation and PLP production, intersubunit complementation |
FEBS letters |
High |
20837012
|
| 2012 |
PDX1 and PfPdx2 interact in vivo in P. falciparum intraerythrocytic parasites, demonstrated by pull-down of transgenic parasites overexpressing both proteins. Overexpression of the PDX1/PfPdx2 complex raises intracellular PLP levels from 12.5 µM (wild-type) to 36.6 µM and confers increased tolerance to singlet oxygen (¹O₂). Dominant-negative inactive mutants of PDX1 and PfPdx2 rendered parasites susceptible to ¹O₂, establishing a role for PDX1-mediated PLP biosynthesis in antioxidant defence. |
In vivo pull-down from transgenic parasites, live-cell fluorescence microscopy, PLP quantification, dominant-negative mutagenesis |
The Biochemical journal |
High |
22242896
|
| 2012 |
The cryo-EM structure of the P. falciparum PLP synthase complex reveals random association of up to 12 PfPdx2 glutaminase subunits to the dodecameric PDX1 core. Plasmodium PLP synthase uniquely organizes into fibers. Crystal structure shows alternative positioning of an α-helix distinguishing open vs. closed conformations upon substrate binding; the pentose substrate is covalently attached at C1, forming a Schiff base with Lys84 of PDX1. A transient ammonia-transfer tunnel between PfPdx2 glutaminase and PDX1 active sites is regulated during catalysis. Mutagenesis defined critical methionines required for complex formation. |
Cryo-EM, X-ray crystallography, site-directed mutagenesis |
Structure (London, England : 1993) |
High |
22244765
|
| 2013 |
D-erythrose 4-phosphate (E4P) inhibits PDX1 enzymatic activity in a dose-dependent manner, likely by interfering with ribose 5-phosphate (R5P) imine bond formations at the active site. The E4P hydrazide analogue 4PEHz selectively inhibits PDX1 with IC50 43 µM (free enzyme) and 16 µM in the heteromeric PLP synthase complex. Transgenic P. falciparum overexpressing PDX1 and PfPdx2 showed protection against 4PEHz, confirming on-target activity in vivo. |
In vitro enzyme inhibition assay, molecular docking, transgenic parasite overexpression protection assay, P. falciparum culture IC50 |
The Biochemical journal |
High |
23039077
|
| 2020 |
In P. vivax, the PDX1 ortholog (Pdx1) forms a dodecameric complex and, when mixed with Pdx2, assembles into a ring-shaped, monodispersed 24-meric (dodecameric Pdx1 core decorated with Pdx2) heteromeric complex, as determined by DLS, SAXS, and EM. Pdx2 alone exists in monomeric and various oligomeric states but forms a monodispersed complex upon mixing with dodecameric Pdx1. |
Dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), electron microscopy (EM) |
International journal of molecular sciences |
Medium |
32825141
|
| 2022 |
DATP 18 (3,5-bis(quinolin-4-ylmethylene)tetrahydro-2H-pyran-4(3H)-one) inhibits PDX1 enzymatic activity with IC50 20.34 µM in vitro, approximately twofold more potent than the previously known inhibitor 4PEHz (IC50 43 µM), establishing PDX1 as the molecular target of this compound class. |
In vitro enzyme inhibition assay |
ChemMedChem |
Low |
36251345
|