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The bacterial metabolic enzyme 1-deoxy-d-xylulose-5-phosphatesynthase （DXPS） catalyzes the thiamin diphosphate （ThDP）-dependentformation of DXP from pyruvate and d-glyceraldehyde-3-phosphate（d-GAP）. DXP is an essential bacteria-specific metabolitethat feeds into the biosynthesis of isoprenoids, pyridoxal phosphate（PLP）, and ThDP. DXPS catalyzes the activation of pyruvate to givethe C2α-lactylThDP （LThDP） adduct that is long-lived on DXPSin a closed state in the absence of the cosubstrate. Binding of d-GAP shifts the DXPS-LThDP complex to an open state which coincideswith LThDP decarboxylation. This gated mechanism distinguishes DXPSin ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate,while other pyruvate decarboxylases readily activate LThDP for decarboxylation,is a long-standing question in the field. We propose that an activesite network functions to prevent LThDP activation on DXPS until thecosubstrate binds. Binding of d-GAP coincides with a conformationalshift and disrupts the network causing changes in the active sitethat promote LThDP activation. Here, we show that the substitutionof putative network residues, as well as nearby residues believedto contribute to network charge distribution, predictably affectsLThDP reactivity. Substitutions predicted to disrupt the network havethe effect to activate LThDP for decarboxylation, resulting in CO2 and acetate production. In contrast, a substitution predictedto strengthen the network fails to activate LThDP and has the effectto shift DXPS toward the closed state. Network-disrupting substitutionsnear the carboxylate of LThDP also have a pronounced effect to shiftDXPS to an open state. These results offer initial insights to explainthe long-lived LThDP intermediate and its activation through disruptionof an active site network, which is unique to DXPS. These findingshave important implications for DXPS function in bacteria and itsdevelopment as an antibacterial target.
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