Bisubstrate Analog Inhibitors of DXP Synthase Show Species Specificity.

1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) is a unique thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the formation of DXP, a branchpoint metabolite required for the biosynthesis of vitamins and isoprenoids in bacterial pathogens. DXPS has relaxed substrate specificity and utilizes a gated mechanism, equipping DXPS to sense and respond to diverse substrates. We speculate that pathogens utilize this distinct gated mechanism in different ways to support metabolic adaptation during infection. DXPS is susceptible to time-dependent inhibition by bisubstrate analogs. We suggest that potential differences in the ligand-gated mechanism that may accompany alternative activities of DXPS homologues may enable the development of species-specific bisubstrate analog inhibitors. Here, we evaluate known bisubstrate analog inhibitors of DXPS (DXPS) against DXPS from (DXPS), a Gram-negative pathogen with a remarkable capacity to adapt to diverse environments. Our results indicate that these inhibitors are significantly less potent against DXPS compared to DXPS. Acceptor site residues that stabilize the phosphonolactyl-ThDP adduct (PLThDP) of bisubstrate analog d-PheTrAP on DXPS are not as critical for stabilization of this PLThDP adduct on DXPS. Substitution of R99 or the analogous R106 reduces the potency of both d-PheTrAP and the simpler BAP scaffold, suggesting a common role of these arginine residues in stabilizing PLThDP adducts. However, although R99 is required for potent, time-dependent inhibition of DXPS by d-PheTrAP, R106 does not appear to govern slow-onset inhibition. This work demonstrates that species-specific targeting of DXPS by bisubstrate analogs is possible and highlights mechanistic differences that should be considered in the design of homologue-specific inhibitors, toward narrow-spectrum approaches targeting DXPS.