Observation of Thiamin-Bound Intermediates and Microscopic Rate Constants for Their Interconversion on 1-Deoxy-d-xylulose 5-Phosphate Synthase: 600-Fold Rate Acceleration of Pyruvate Decarboxylation by d-Glyceraldehyde-3-phosphate.

Abstract

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.

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

“…42,43 The reaction commences with the typical ThDP-catalyzed pyruvate decarboxylation, and LThDP was found to be remarkably stable in the absence of d -GAP. 23 The principal results on DXPS are the following.…”

“…23 DXPS complexed with ThDP titrated with pyruvate (100–1000 µM) at 5 °C displayed a positive CD band at 312 nm, suggesting formation of the 1′,4′-iminopyrimidinylLThDP intermediate (Figure 10A). 23 These two CD bands were not apparent when apo-DXPS was complexed with MeOThDP (Figure 10B), indicating that MeOThDP has a different binding mode that does not give rise to the CD signatures for different forms of ThDP. Similar observations were made on the Arg478Ala variant of DXPS [for which an activity of 0.059 ± 0.007 µmol min −1 (mg of Arg478Ala DXPS) −1 was determined] (Figure S7).…”

“…To calculate the DXPS activity and the corresponding rate constants, the recently reported molar ellipticity of DXP of 13200 deg cm 3 dmol −1 was used. 23 …”

Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

“…This assay still involved an additional step for derivatization of the product. Recently, an assay based on circular dichroism has been reported for DXS (51). This assay appeared to be extremely important for studying the mechanistic behavior of DXS, illustrating detailed insights about different TPP-bound intermediates involved in the DXS-catalyzed reaction.…”

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.…”