Chiu, A.; Liu, L.; Seibold, S.; Battaile, K.; Craig, J.; Harmon, E.; Subramanian, S.; Chakafana, G.; Early, J.; Cron, L.; Staker, B.; Myler, P. J.; Lovell, S. J.; Van Voorhis, W.; Asojo, O.

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-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 TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and preliminary structure-functional analyses of three crystal structures of TvPPi-PFK are presented. All three structures organize as tetramers with the conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. Comparative analysis with structural neighbors from other organisms demonstrated that despite sharing <29% sequence identity, TvPPi-PFK's protomer shares overall topology with both PPi- and ATP-dependent PFKs. Mass photometry confirmed that TvPPi-PFK formed tetramers under near-physiological conditions. Unexpectedly, TvPPi-PFK crystals dephosphorylate ATP to AMP during soaking. In all three structures, either ATP or AMP is bound at the enzyme's dimer interface, typical of ATP-PFKs, but a novel finding for PPi-PFKs. Furthermore, a sugar phosphate binding site was observed in proximity to the ATP-binding site. Thus, the three reported TvPPi-PFK structures validate its established PPi-dependent activity while revealing previously unreported ATP and sugar phosphate binding. This study also lays a foundation for future research into putative ATP-dependent activity of TvPPi-PFK and for evaluating known phosphofructokinase inhibitors as potential therapeutics for trichomoniasis. These findings expand our understanding of PFK superfamily diversity and support the continued exploration of TvPPi-PFK as a drug target for trichomoniasis.