Thiamin Diphosphate Activation in 1-Deoxy-d-xylulose 5-Phosphate Synthase: Insights into the Mechanism and Underlying Intermolecular Interactions

Abstract

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4'-aminopyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4'-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ∼10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes induced by conformational variations in the active site. Additionally, mutagenesis studies reveal DXS to be an evolutionarily resilient enzyme. Particularly, we hypothesize that residues H82 and H304 may act in a compensatory fashion if the other is lost due to mutation. Further, nucleus-independent chemical shifts (NICSs) and aromatic stabilization energy (ASE) calculations suggest that reduction in TDP aromaticity also serves as a factor for regulating ylide formation and controlling reactivity.

Figure 1

Schematic of isoprene production via MVA or MEP pathway. MVA pathway produces DMADP via a secondary enzyme, IDP isomerase.^, MEP pathway directly synthesizes both isoprene molecules.

Figure 2

Proposed general mechanism for DXP biosynthesis. Pieces of each step are labeled with different colors to indicate where they originate from. Red represents pyruvate, and blue represents the pieces affiliated with G3P.

Figure 3

Structure and relationship of the four possible tautomeric/ionization states proposed for the cofactor of TDP dependent enzymes.^,– Key atoms have been given names for reference purposes throughout this article.

Figure 4

Representations of the RS for DHM (a) and WMM (b). The dashed black lines illustrate the proton transfer reaction.

Figure 5

Minimum energy profiles computed for the WMM and DHM. The different x-axes are used because of differences in the reaction coordinate ranges for WMM vs DHM; both are associated with the same y-axis. The ΔE^‡ are 22.7 kcal·mol⁻¹ and 33.7 kcal·mol⁻¹ for the WMM (gray circles) and DHM (black squares), respectively.

Figure 6

Representative conformational changes between the RS (yellow) and TS (green) of the DHM.

Figure 7

Illustration of the proton transfer from E373 to TDP’s AP ring during the tautomerization reaction. (a) and (b) represent the reactant and product states, respectively. While this figure only depicts the structures of the WMM, a similar response was observed during the DHM.

Figure 8

Active site conformation of the residues discussed in the CPA results. Images show both the RS (yellow) and TS (green). (a) illustrates the DHM, while (b) shows the WMM.

Figure 9

Illustrated above are the computed RS (yellow) and TS (green) dipoles of the WMM and DHM. (a) is the DHM, and (b) is the WMM.

Figure 10

Analysis for 18 ns of the unrestrained simulation of the 2O1X DXS structure utilized in this investigation. (a) shows the distances over time for N_ε of H434 to N4′ of TDP’s amino group. (b) shows the fluctuations for backbone (black), and side chains (gray) for residues H51, K101, H124, K289, E373, D430, and H434. These residues represent the QM region and key CPA residues previously discussed. (c) is a snapshot from the 18 ns trajectory with H434 in proximity to the 4′-amino group. (d) is representative of H434 in the second conformation.

Figure 11

Homodesmotic reaction used in evaluating the aromatic stabilization energy for a model TDP.