Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase ( Tv PPi-PFK)
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Abstract
Pyrophosphate-dependent phosphofructokinases provide an alternative route through central carbon metabolism by using inorganic pyrophosphate rather than ATP to phosphorylate fructose-6-phosphate. These enzymes are absent from mammals but occur in several anaerobic and parasitic organisms, where they may support metabolic flexibility and represent potential therapeutic vulnerabilities. Here, we report three crystal structures of Trichomonas vaginalis PPi-dependent phosphofructokinase 1 (TvPPi-PFK), a cytosolic enzyme that contributes substantially to parasite glycolytic flux. The structures show that TvPPi-PFK adopts the conserved two-domain PFK fold, assembles as a tetramer, and preserves the canonical PPi/MgPPi²⁻-binding motifs required for pyrophosphate-dependent catalysis. Mass photometry confirms that TvPPi-PFK is predominantly tetrameric in solution and shows that nucleotide- and Mg²⁺/PPi-containing conditions further stabilize the tetrameric assembly. Unexpectedly, the ligand-bound structures reveal ATP, AMP, and sugar-phosphate ligands at a recurring dimer-interface site that is spatially distinct from the canonical PPi/MgPPi²⁻ catalytic donor-binding pocket. This non-canonical interface is more accessible in TvPPi-PFK than in available PPi-PFK structures, suggesting a previously unrecognized ligand-recognition feature. These findings expand the structural landscape of the PFK superfamily and provide a framework for testing how nucleotide and sugar-phosphate binding may influence TvPPi-PFK function, regulation, and future antitrichomonal drug-discovery efforts.
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Abstract
Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase ( Tv PPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. Tv PPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by Tv PPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis . The purification, crystallization, structure determination, and crystal structures of Tv PPi-PFK are reported. Tv PPi-PFK is the first reported eukaryotic PPi-PFK structure. Tv PPi-PFK retains the overall PPi-PFK topology observed in bacterial PPi-PFK including conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. In addition to the catalytic PPi-PFK binding sites, Tv PPi-PFK has two additional ligand binding sites. The first binds AMP usurped during protein production and helps stabilize the Tv PPi-PFK tetramer. A second ligand binding site was observed in proximity to the AMP-binding site and accommodates sugar phosphates soaked into preformed crystals. This sugar phosphates binding site is distinct from the Tv PPi-PFK active site that binds fructose-6-phosphate. Future mutagenesis and activity studies are planned to determine the relevance of both sites.
Synopsis
The production, crystallization, and crystal structures of a pyrophosphate-dependent phosphofructokinase from Trichomonas vaginalis ( Tv PPi-PFK) are reported. Tv PPi-PFK has a prototypical PPi-PFK active site as well as unexpected AMP and sugar-phosphate binding sites at the dimer interface.
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- europepmc
- last seen: 2026-05-20T01:45:00.602351+00:00
- unpaywall
- last seen: 2026-09-10T06:36:06.991349+00:00
License: CC-BY-NC-ND-4.0