Defining critical residues for substrate binding to 1‐deoxy‐d‐xylulose 5‐phosphate synthase – active site substitutions stabilize the predecarboxylation intermediate C2α‐lactylthiamin diphosphate

Abstract

1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner, and is the first step in the essential pathway to isoprenoids in human pathogens. Understanding the mechanism of this unique enzyme is critical for developing new anti-infective agents that selectively target isoprenoid biosynthesis. The present study uses mutagenesis and a combination of protein fluorescence, circular dichroism and kinetics experiments to investigate the roles of Arg-420, Arg-478 and Tyr-392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism and predict Arg-420 and Arg-478 are involved in binding of the acceptor substrate, d-GAP. d-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg-420 and Arg-478, also accelerates decarboxylation of the pre-decarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating this binding interaction is not absolutely required, and the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr-392 contributes to d-GAP affinity and is not required for LThDP formation or its d-GAP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate LThDP is significantly stabilized on R420A and Y392F variants compared to wild type DXP synthase in the absence of acceptor substrate, yet these substitutions do not appear to impact the rate of d-GAP-promoted LThDP decarboxylation in the presence of high d-GAP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues to promote d-GAP binding which in turn facilitates decarboxylation, and further highlight interesting differences between DXP synthase and other ThDP-dependent enzymes.

Competence of Thiamin Diphosphate-Dependent Enzymes with 2′-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin

“…Earlier, it was reported that Arg478 is important for GAP binding and its recognition, but not crucial for catalysis through LThDP formation. 9 Again, the characteristic negative CD band at 320 nm was not apparent on MeOThDP binding (Figure S7). Upon titration with donor substrate pyruvate, the positive CD band at 313 nm assigned earlier to LThDP was also evident for the Arg478Ala DXPS complexed with ThDP, but not with MeOThDP (Figure S7).…”

“…9 The assay mixture (2.4 mL) contained 50 mM KH 2 PO 4 , 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 0.10 mM ThDP (0.10 mM MeOThDP), 2.0 mM MgCl 2 , 1.0 mM pyruvate, 1.0 mM DTT, 1 mM GAP, and 1% glycerol. The reaction was started by addition of 0.02 mg of DXPS, and the spectrum was recorded for 400 s at 37 °C.…”

Progress in the experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations

“…CD experiments, complemented with the TH method to assist the CD assignments, revealed that formation of LThDP is rate-limiting overall in the sequence of reactions carried out by DXPS [63,64]. However, LThDP is remarkably stable on the enzyme until the acceptor D-GAP is added.…”

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase

“…Structural studies of DXP synthase indicate the enzyme possesses unique domain architecture 17 and an unusually large active site volume; 18 these observations are consistent with mechanistic studies offering support for ternary complex formation in DXP synthase catalysis. 19−22 Our mechanistic studies suggest GAP serves two distinct roles in DXP formation, as a trigger for lactyl thiamin diphosphate (LThDP) decarboxylation and as an acceptor substrate during carboligation. 21,22 As new insights emerge about the distinctive characteristics of DXP synthase, so will new opportunities for the development of selective inhibitors.…”