Aromatic residues in mobile regions distal to the active site support the closed conformation of E. coli DXPS

The essential bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is absent in humans, making the enzyme an attractive antimicrobial target. Its product DXP sits at a metabolic branchpoint and is required for biosynthesis of pyridoxal phosphate (PLP), thiamin diphosphate (ThDP), and isoprenoids. DXP is formed via decarboxylation of pyruvate and subsequent carboligation with d-glyceraldehyde-3-phosphate (d-GAP) in a ThDP-dependent manner. In the current mechanistic model, DXPS follows a ligand-gated mechanism. Pyruvate reacts with ThDP to form C2α-lactylThDP (LThDP) which coincides with a shift to a closed conformation. The flexible “spoon” and “fork” motifs become ordered and the catalytic residue H299 is placed within the active site, supporting LThDP formation and persistence in the closed conformation of the E-LThDP complex until binding of d-GAP. We aim to understand the molecular basis for stabilization of the E-LThDP complex in its closed conformation. We propose the conserved aromatic residues Y288, F298, and F304 in the E. coli DXPS spoon and fork motifs form a cluster upon transition from the open to closed form, positioning H299 within the active site where it has roles in LThDP formation and persistence. We conducted mutagenesis studies to elucidate the roles of Y288, F298, and F304 in conformational cycling and catalysis. Variants 1) adopted open conformations, 2) displayed significantly reduced kcat, 3) promoted LThDP decarboxylation, and 4) exhibited decreased affinity for the post-decarboxylation intermediate. Our results support a model in which conserved aromatic residues within the mobile, sequence-diverse spoon/fork motifs promote the closed conformation, supporting catalysis and LThDP persistence.