Toward Understanding the Role of an Active Site Network in LThDP Stabilization on DXPS

Abstract

Microbial metabolic pathways may provide novel, bacteria‐specific targets for antibiotic development, thus providing new strategies to combat the evolving conflict of antibiotic resistance. The bacterial metabolic enzyme 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXPS), which is absent in humans, catalyzes the thiamine diphosphate (ThDP)‐dependent formation of DXP from pyruvate and glyceraldehyde‐3‐phosphate (GAP). DXPS has emerged as an appealing target due to its essentiality in bacterial pathogens, as DXP feeds into the biosynthesis of isoprenoids, ThDP, and pyridoxal phosphate (PLP), positioning DXPS as a central player in bacterial metabolism. DXPS has a unique mechanism in ThDP enzymology, in which binding of a small molecule “trigger” is required to induce a conformational change from the closed to an open conformation, essential for decarboxylation of the first enzyme bound intermediate, C2α−lactyl‐ThDP (LThDP). GAP is one such trigger molecule, likely binding at a site that is distinct from its acceptor binding site. However, how DXPS stabilizes LThDP prior to GAP‐induced decarboxylation remains unknown. In this study, we propose an active site network connects key active site histidine residues to induce the closed conformation upon reaction with pyruvate, stabilizing the LThDP intermediate. E. coli Arginine 99 may be an essential component of this network. We found that artificial disruption of this network through substitution of R99 (R99A) 1) increases KmGAP three‐fold, 2) destabilizes LThDP (detected by Circular Dichroism), and 3) shifts the conformation to the open state believed to induce decarboxylation (detected by HDX‐MS and limited proteolysis). Overall, the results support the hypothesis that R99 serves to maintain the closed conformation by connecting the key histidine residues, providing preliminary evidence for the existence of an active site network that links catalysis to conformational dynamics. Targeting the unique gated mechanism of DXPS could prove to be an effective strategy for developing a selective antibiotic that has far reaching impacts on bacterial metabolism in a pathogenic state.