The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-D-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as 2-hydroxyethyl donor with D-glyceraldehyde-3-phosphate (D-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and pre-steady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound pre-decarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of D-GAP, while addition of D-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and D-GAP bound, and the central enzyme-bound enamine reacts with D-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved CD spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of D-GAP suggests a new approach to inhibitor design.

The six-transmembrane protein glycerophosphodiester phosphodiesterase 2 (GDE2) induces spinal motor neuron differentiation by inhibiting Notch signaling in adjacent motor neuron progenitors. GDE2 function requires activity of its extracellular domain that shares homology with glycerophosphodiester phosphodiesterases (GDPD). GDPDs metabolize glycerophosphodiesters into glycerol-3-phosphate and corresponding alcohols but whether GDE2 inhibits Notch signaling by this mechanism is unclear. Here, we show that GDE2, unlike classical GDPDs, cleaves glycosylphosphatidylinositol (GPI)-anchors. GDE2 GDPD activity inactivates the Notch activator RECK by releasing it from the membrane by GPI-anchor cleavage. RECK release disinhibits ADAM protease-dependent shedding of the Notch ligand Delta-like 1 (Dll1) leading to Notch inactivation. This study identifies a previously unrecognized mechanism to initiate neurogenesis that involves GDE2 mediated surface cleavage of GPI-anchored targets to inhibit Dll1-Notch signaling.

Background: 1-Deoxy-D-xylulose 5-phosphate (DXP) synthase is a thiamine diphosphate (ThDP)-dependent enzyme in pathogen isoprenoid biosynthesis and a potential drug target. Results: Tryptophan fluorescence and kinetic analyses show that donor and acceptor substrates bind reversibly and independently to DXP synthase. Conclusion: DXP synthase catalyzes a novel, ThDP-dependent, random sequential mechanism. Significance: Targeting the unique kinetic mechanism of DXP synthase could lead to new anti-infective agents.

We study the deposition of line segments on a two-dimensional square lattice. The estimates for the coverage at jamming obtained by Monte-Carlo simulations and by 7 th -order time-series expansion are successfully compared. The non-trivial limit of adsorption of infinitely long segments is studied, and the lattice coverage is consistently obtained using these two approaches.

TLC is used extensively in nucleic acid chemistry to monitor the progress of chemical reactions, to assay fractions collected from a larger chromatographic separation (e.g., column chromatography), and to determine optimal conditions prior to column chromatography. This unit describes methods for spotting test compounds onto a TLC plate, developing the plate in a suitable solvent system, visualizing the results, and calculating the retention factor (R(f)). Candidate compounds can be co-spotted for identification without relying on R(f) values.

DXP synthase catalyzes the formation of 1-deoxy-D-xylulose 5-phosphate, an essential precursor in pathogen isoprenoid biosynthesis. The selective inhibition of this ThDP-dependent transformation is a challenging goal in the development of isoprenoid biosynthesis inhibitors. Potent, selective inhibitors could lead to new anti-infective agents. Here, we demonstrate selective inhibition of E. coli DXP synthase by butylacetylphosphonate.

Edited by Joseph M. Jez This work was supported by National Institutes of Health Grants R35 GM126982 (to C. L. D.) and R01 GM084998 (C. L. F. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1-S13 and Tables S1-S4. The atomic coordinates and structure factors (codes 6OUV and 6OUW) have been deposited in the Protein Data Bank (http://wwpdb.org/).

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.