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

Abstract 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 between the 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 C2⍺-lactylThDP (LThDP) which coincides with a shift to a closed conformation. The flexible “spoon” and “fork” motifs become ordered, situating the catalytic residue H299 within the active site which supports LThDP persistence and the closed conformation of the E-LThDP complex until binding of D-GAP. Our goal is to understand the molecular basis for stabilization of the E-LThDP complex in its closed conformation in the absence of D-GAP. 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 to position H299 within the active site, necessary for LThDP persistence. Here, we conducted mutagenesis studies to elucidate the roles of Y288, F298, and F304 in conformational cycling and catalysis. On each variant, the conformational equilibrium favored an open state, hindered intermediate formation and persistence, and promoted intermediate release from the active site. Our results support a model in which conserved aromatic residues within the mobile, sequence-diverse spoon and fork motifs promote the closed conformation and support catalysis. Competing Interest Statement The authors have declared no competing interest. Footnotes In this revision we corrected kinetic calculations, added a supplemental figure, and edited text for completeness and grammatical accuracy. Abbreviations - DXPS - 1-deoxy-D-xylulose 5-phosphate synthase - D-GAP - D-glyceradehyde-3-phosphate - MAP - methyl acetylphophonate - ThDP - thiamin diphosphate - LThDP - C2⍺-lactyl-thiamin diphosphate - HEThDP - 2-(1-hydroxyethyl)thiamine diphosphate - PLThDP - C2⍺-phosphonolactylthiamin diphosphate - HDX-MS - hydrogen-dueterium mass spectrometry - AP - aminopyrimidine tautomer of ThDP - IP - 1’,4’-iminopyrimidine tautomer of ThDP