Conformational dynamics of 1-deoxy- d -xylulose 5-phosphate synthase on ligand binding revealed by H/D exchange MS

Abstract

The enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-d-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B) and pyridoxal (vitamin B). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by d-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen-deuterium (H/D) exchange MS (HDX-MS) of full-length DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. () The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183-238 and 292-317) not observed by X-ray crystallography. () Three regions of DXPS (spanning residues 42-58, 183-199, and 278-298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. () The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

“…9A) suggesting that the PDZ domains may undergo only local conformational changes upon pSer 290 dephosphorylation. Consistent with previous observations in other proteins (64,65), and as illustrated in Fig. 9B (panels ii and iv), the regions exhibiting conformational changes upon pSer 290 dephosphorylation display a typical bimodal distribution of deuterium uptake as typified by the NHERF1 (39 -59) peptide.…”

Oxidative decarboxylation of pyruvate by 1-deoxy-d-xyulose 5-phosphate synthase, a central metabolic enzyme in bacteria

“…D-GAP or O 2 ) to trigger efficient LThDP decarboxylation on DXP synthase is established, the mechanism by which this occurs is not fully elucidated. Several mechanistic studies of DXP synthase have revealed compelling evidence for D-GAP-dependent conformational changes (12,16,17). For example, a recent HDX-MS study showed that a donor substrate analog and D-GAP induce closed and open conformations of DXP synthase, respectively (12).…”

“…Several mechanistic studies of DXP synthase have revealed compelling evidence for D-GAP-dependent conformational changes (12,16,17). For example, a recent HDX-MS study showed that a donor substrate analog and D-GAP induce closed and open conformations of DXP synthase, respectively (12). It is conceivable that D-GAP binding induces a conformational change that destabilizes the LThDP intermediate, lowering the barrier for chemical decarboxylation of LThDP (12,15,16).…”

“…For example, a recent HDX-MS study showed that a donor substrate analog and D-GAP induce closed and open conformations of DXP synthase, respectively (12). It is conceivable that D-GAP binding induces a conformational change that destabilizes the LThDP intermediate, lowering the barrier for chemical decarboxylation of LThDP (12,15,16). Considering the proposal that 2-oxoacid decarboxylation is reversible on ThDP-dependent enzymes (31)(32)(33), it is also possible that an equilibrium between LThDP and the C2␣-carbanion/CO 2 is established on DXP synthase in a conformation that limits release of CO 2 from the enzyme.…”

Development of Software for the In-Depth Analysis of Protein Dynamics as Determined by MALDI Mass Spectrometry-Based Hydrogen/Deuterium Exchange

“…[15][16][17] Furthermore, the shapes of the isotopic distribution curves in HDX contain crucial information on the multiple conformations of a protein. [21][22][23] Consequently, a dedicated so ware program for the highly precise analysis of isotopic distribution would be expected to dramatically enhance investigations of the structural equilibrium between multiple protein conformations. ere is, however, no so ware currently available for analyzing the protein conformation and dynamics MALDI HDX MS data except for TOF2H.…”