| 2004 |
PDX2 (PfPdx2) acts as the glutaminase subunit of the P. falciparum PLP synthase complex, functioning together with PfPdx1 to catalyze 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 auxotroph yeast cells confirmed involvement of both proteins in de novo vitamin B6 biosynthesis. |
Recombinant protein biochemistry (glutaminase activity assay), yeast complementation assay |
The Journal of biological chemistry |
High |
15590634
|
| 2005 |
PDX2 functions as the glutaminase subunit of the heteromeric PLP synthase complex; complex formation with Pdx1 is required for catalytic activity of either subunit. The 1.62 Å crystal structure of PfPdx2 reveals an obstructed oxyanion hole indicating a resting state that is activated upon Pdx1-Pdx2 complex formation. A chimeric bi-enzyme of Pdx1 with bacterial YaaE (a Pdx2 ortholog) forms in vivo and in vitro but does not attain full catalytic activity, emphasizing species-specific structural requirements for productive interaction. |
X-ray crystallography (1.62 Å structure of PfPdx2), in vitro reconstitution, glutaminase activity assay, in vivo/in vitro chimera formation |
The Journal of biological chemistry |
High |
16339145
|
| 2007 |
Thermodynamic characterization by isothermal titration calorimetry showed that l-glutamine binding increases the affinity of PDX2 for Pdx1 approximately 30-fold and alters the thermodynamic signature of complex formation, indicating substrate-dependent tightening of the PDX2–Pdx1 interface. The complex interface is dominated by hydrophobic interactions (negative heat capacity). Swapping the αN-helix of PfPdx1 with the corresponding B. subtilis segment enabled cross-binding to B. subtilis Pdx2 and partially elicited glutaminase activity in BsPdx2, demonstrating that catalytic activation of PDX2 is mechanistically linked to formation of the Pdx1 αN-helix interface. |
Isothermal titration calorimetry, swap-mutant biochemistry, glutaminase activity assay, 3D homology modelling |
Journal of molecular biology |
High |
17950752
|
| 2008 |
Mutational analysis of P. falciparum Pdx1 interface residues (R85, H88, E91 at the Pdx1:Pdx1 interface) showed that their mutation to alanine impedes both Pdx1 activity and PDX2 binding, establishing that Pdx1 self-assembly is a prerequisite for PDX2 recruitment. Mutation of active-site residues D26, K83, K151 blocked enzyme activity but did not prevent PDX2 binding or PDX2 glutaminase activity, demonstrating that PDX2 can bind a hexameric Pdx1 scaffold but full catalytic output requires a dodecameric assembly. |
Site-directed mutagenesis, biochemical activity assays, size-exclusion chromatography, co-purification |
PloS one |
High |
18350152
|
| 2009 |
A Glycine-155-to-Alanine mutation in Pdx1 produces a hexameric (not dodecameric) assembly in vitro with significant secondary-structure changes, yet this mutant retains the ability to bind PDX2 and support PDX2 glutaminase activity, demonstrating that PDX2 binding does not require the full dodecameric Pdx1 state, whereas Pdx1 catalytic activity does. |
Static and dynamic light scattering, circular dichroism, co-purification, enzyme activity assays |
Biochimica et biophysica acta |
Medium |
19272411
|
| 2010 |
C-terminal deletion analysis of Pdx1 dissected four distinct functions contributed by the C-terminus: Pdx1 monomer assembly, ribose 5-phosphate binding, formation of reaction intermediate I320, and PLP synthesis. PLP synthesis is the only C-terminal function that can be supplied in trans, explaining the cooperative nature of the PDX2–Pdx1 complex and the cross-talk between subunits. |
C-terminal deletion mutagenesis, enzymatic activity assays, mass spectrometry (intermediate detection) |
FEBS letters |
Medium |
20837012
|
| 2012 |
Cryo-EM analysis revealed that PDX2 glutaminase subunits associate randomly (up to 12 subunits) onto the dodecameric Pdx1 core, and P. falciparum PLP synthase organizes into fibers. Crystal structure of the complex shows that an α-helix adopts an open vs. closed conformation upon substrate binding; the pentose substrate is covalently attached via its C1 in a Schiff base with Lys84 of Pdx1. A transient ammonia transfer tunnel between PDX2 and Pdx1 active sites is regulated during catalysis. Mutagenesis defined critical conserved methionines required for complex function while also showing plasticity in tunnel construction across species. |
Electron microscopy (EM), X-ray crystallography, site-directed mutagenesis, enzymatic assays |
Structure (London, England : 1993) |
High |
22244765
|
| 2012 |
In vivo pull-down experiments from transgenic P. falciparum parasites overexpressing both PfPdx1 and PDX2 confirmed a direct interaction between the two proteins inside the parasite. Overexpression of the complex elevated intracellular PLP levels from 12.5 µM (wild-type) to 36.6 µM and increased tolerance to singlet oxygen (¹O₂). Dominant-negative inactive mutants of either PfPdx1 or PDX2 rendered parasites susceptible to ¹O₂, establishing that de novo PLP synthesis via the PDX2-containing complex is required for singlet oxygen detoxification. |
In vivo pull-down (transgenic parasite overexpression), live-cell fluorescence microscopy, PLP quantification, transcription analysis, dominant-negative mutagenesis |
The Biochemical journal |
High |
22242896
|
| 2013 |
PDX2 assembles with Pdx1 into a heteromeric PLP synthase complex in P. falciparum; transgenic parasite lines overexpressing both PfPdx1 and PDX2 showed protection against the Pdx1 inhibitor 4PEHz (IC50 shift from 16 µM for the complex to reduced sensitivity with extra copies), confirming that the PDX2-containing complex is the in vivo target of the inhibitor and is directly required for PLP biosynthesis in the parasite. |
Transgenic overexpression in P. falciparum, in vitro Pdx1 inhibition assay, parasite growth inhibition assay |
The Biochemical journal |
Medium |
23039077
|
| 2020 |
In P. vivax, PDX2 exists in monomeric and multiple oligomeric states in solution; upon mixing with dodecameric Pdx1, polydisperse PDX2 is converted to a monodispersed heteromeric 24-meric Pdx1–PDX2 (Pdx) complex, as determined by DLS, SAXS (showing a ring-shaped dodecameric Pdx1 decorated with PDX2), and electron microscopy. |
Dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), electron microscopy (EM) |
International journal of molecular sciences |
Medium |
32825141
|