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DXPS synthase uses thiamine diphosphate (ThDP) to convert pyruvate and D-glyceraldehyde 3‑phosphate (D-GAP) into 1-deoxy-D-xylulose 5-phosphate (DXP), an essential bacterial metabolite. DXP is not utilized by humans; hence, DXPS has been an attractive antibacterial target. Here, we investigate DXPS from Deinococcus radiodurans ( Dr DXPS), showing that it has similar kinetic parameters K m d-GAP and K m pyruvate (54 ± 3 µM and 11 ± 1 µM, respectively) and comparable catalytic activity ( k cat = 45 ± 2 min -1) to previously studied bacterial DXPS enzymes, and employing it to obtain missing structural data on this enzyme family. In particular, we have determined crystallographic snapshots of Dr DXPS in two states along the reaction coordinate - a structure of Dr DXPS bound to C2α-phosphonolactylThDP (PLThDP), mimicking the native pre-decarboxylation intermediate C2α-lactylThDP (LThDP), and a native post-decarboxylation state with a bound enamine intermediate. The 1.94-Å resolution structure of PLThDP-bound Dr DXPS delineates how two active site histidine residues stabilize the LThDP intermediate. Meanwhile, the 2.40-Å resolution structure of an enamine intermediate-bound Dr DXPS reveals how a previously unknown 17-Å conformational change removes one of the two histidine residues from the active site, likely triggering LThDP decarboxylation to form the enamine intermediate. These results provide insight into how the bi-substrate enzyme DXPS limits side reactions by arresting the reaction on the less reactive LThDP intermediate when its co-substrate is absent. They also offer a molecular basis for previous low-resolution experimental observations that correlate decarboxylation of LThDP with protein conformational changes.