PFK9

PFK9 (PF3D7_0915400) is the ATP-dependent 6-phosphofructokinase of Plasmodium falciparum and a principal control point of parasite glycolysis, catalyzing phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate (PMID:25580350). Despite carrying plant-like PPi-PFK domain features, recombinant PFK9 and parasite extracts display ATP-dependent activity with no detectable PPi-dependent activity, establishing it as a catalytically active, ATP-utilizing enzyme; the orthologous enzyme in P. berghei shows atypical allosteric behavior in which phosphoenolpyruvate activates rather than inhibits and AMP activates only marginally (PMID:25580350). PFK9 exerts substantial control over glycolytic flux, and modulating its activity redistributes carbon between upper glycolysis, the pentose phosphate pathway, and the MEP isoprenoid pathway: hypomorphic PFK9 mutations suppress the metabolic dysregulation caused by loss of HAD2, while other PFK9 mutations confer resistance to the glycolytic inhibitor PS-3 with reduced upper-glycolytic flux, lower ATP, and diminished MEP intermediates (PMID:30425143, PMID:33293381). This positions PFK9 as a node of metabolic plasticity linking glycolytic flux to downstream isoprenoid synthesis (PMID:30425143, PMID:33293381). Beyond its glycolytic role, PFK9 has been identified as a candidate substrate for phosphorylation by PfCDPK4 during male gametogenesis and detected in physical proximity to the artemisinin-resistance protein K13, but neither association has been functionally characterized in the available corpus (PMID:34724830, PMID:33095255).

1. 2014 Medium

   Whether the Plasmodium enzyme with plant-like PPi-PFK domain features actually uses ATP or inorganic pyrophosphate was resolved by direct enzymatic reconstitution, defining PFK9 as a genuine ATP-dependent phosphofructokinase.

   Evidence In vitro enzymatic assay of recombinant PFK9 and parasite extracts with ATP vs. PPi substrates; allosteric regulation assays in the P. berghei ortholog

   PMID:25580350

   Open questions at the time

   • No structural model of the P. falciparum enzyme reported
   • Allosteric data are from the P. berghei ortholog, not P. falciparum PFK9 directly
   • Physiological regulators controlling PFK9 activity in situ not defined
2. 2016 Low

   Whether phosphofructokinase is merely one glycolytic enzyme or a rate-controlling step was addressed by kinetic modelling, predicting a high flux control coefficient for the reaction PFK9 catalyzes.

   Evidence Detailed kinetic mathematical modelling of P. falciparum glycolysis with flux control coefficient sensitivity analysis

   PMID:26648082

   Open questions at the time

   • Computational prediction only, no direct in vitro or in vivo flux perturbation of PFK9
   • Does not identify which parameters set the control coefficient
   • Model not validated against a PFK9 knockdown or mutant
3. 2018 Medium

   Whether PFK9 activity is a tunable node connecting glycolysis to downstream metabolism was tested genetically, showing that reducing PFK9 function suppresses HAD2-loss dysregulation and remodels glycolytic and MEP intermediate levels.

   Evidence Forward genetic suppressor screen with whole-genome sequencing of suppressor mutants and metabolic profiling of glycolytic and MEP pathway intermediates

   PMID:30425143

   Open questions at the time

   • Precise biochemical impact of the hypomorphic mutations on PFK9 kinetics not measured
   • Mechanism linking glycolytic flux to MEP intermediates not resolved at enzyme level
   • Single lab
4. 2020 Medium

   Whether PFK9 governs the partitioning of carbon among competing metabolic routes was addressed by drug-resistance selection, linking PFK9 mutations to reduced upper-glycolytic flux, lower ATP, increased pentose phosphate flux, and reduced MEP intermediates.

   Evidence In vitro PS-3 resistance selection with whole-genome sequencing, metabolic flux analysis, and metabolite profiling

   PMID:33293381

   Open questions at the time

   • Whether resistance is due to altered PFK9 catalysis versus regulation not distinguished
   • Direct enzymatic characterization of resistant PFK9 alleles absent
   • Single lab
5. 2021 Low

   Whether PFK9 is subject to signaling-driven post-translational control during sexual development was probed phosphoproteomically, identifying PFK9 as a candidate PfCDPK4 substrate in male gametogenesis.

   Evidence Phosphoproteomics of cdpk4 knockout vs. wild-type gametocytes plus in vitro kinase assay with recombinant PfCDPK4 and a synthetic PFK9-derived peptide

   PMID:34724830

   Open questions at the time

   • In vitro peptide assay does not demonstrate phosphorylation of full-length PFK9 in situ
   • Functional consequence of phosphorylation for PFK9 activity or gametogenesis unknown
   • Phosphosite not mapped in the intact protein context
6. 2021 Low

   Whether PFK9 physically associates with the artemisinin-resistance determinant K13 was raised by interactome data, detecting PFK9 as a shared Co-IP and BioID interactor of K13.

   Evidence Co-immunoprecipitation with anti-K13 monoclonal antibody and cross-referenced BioID proximity labelling, reported in a review

   PMID:33095255

   Open questions at the time

   • Single Co-IP/BioID detection without reciprocal validation or functional testing
   • Direct versus proximal/indirect association not distinguished
   • Biological role of any PFK9–K13 association undefined

• How PFK9 activity is dynamically regulated in vivo — the functional consequences of PfCDPK4 phosphorylation, the significance of the K13 association, and the structural basis of its atypical allosteric behavior — remains unresolved.
• No structural model or full-length in situ phosphorylation data
• PFK9–K13 interaction lacks functional validation
• Enzymatic characterization of disease/resistance-associated PFK9 alleles missing