Conformational dynamics of the flexible catalytic loop in Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase

Abstract

In mycobacteria, the biosynthesis of the precursors to the essential isoprenoids, isopentenyl diphosphate and dimethylallyl pyrophosphate is carried out by the methylerythritol phosphate pathway. This route of synthesis is absent in humans, who utilize the alternative mevalonate acid route, thus making the enzymes of the methylerythritol phosphate pathway of chemotherapeutic interest. One such identified target is the second enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Only limited information is currently available concerning the catalytic mechanism and structural dynamics of this enzyme, and only recently has a crystal structure of Mycobacterium tuberculosis species of this enzyme been resolved including all factors required for binding. Here, the dynamics of the enzyme is studied in complex with NADPH, Mn2+, in the presence and absence of the fosmidomycin inhibitor using conventional molecular dynamics and an enhanced sampling technique, reversible digitally filtered molecular dynamics. The simulations reveal significant differences in the conformational dynamics of the vital catalytic loop between the inhibitor-free and inhibitor-bound enzyme complexes and highlight the contributions of conserved residues in this region. The substantial fluctuations observed suggest that 1-deoxy-D-xylulose 5-phosphate reductoisomerase may be a promising target for computer-aided drug discovery through the relaxed complex method.

Figure 1

Crystal structure of asymmetric homodimer of MtDXR (PDB ID: 2JCZ). Monomer A has been coloured according to the following structural features: blue: N-terminal, red: C-terminal, orange: catalytic domain, yellow: extended loop of catalytic domain, purple: catalytic loop in closed conformation. NADPH is shown bound to the N-terminal and fosmidomycin bound in the active site of the catalytic domain close to Mn²⁺ (van der Waals representation). Monomer B is coloured in cyan with the catalytic loop in an open conformation.

Figure 2

RMSD of Cα atoms from the equilibrated starting structure over the length of MD1 simulations of DXR–MN (red line) and for DXR–FMN (black line). Results of the other simulations were observed to be similar and are not shown here

Figure 3

Average RMSF of Cα atoms of monomer A calculated from (A) 12 RDFMD simulations of DXR–MN, (B) 2 MD simulations of DXR–MN (C) 11 RDFMD simulations of DXR–FMN and (D) 2 MD simulations of DXR–FMN.

Figure 4

Sampling of clusters over 40 ns MD (MD1 and MD2) and 4.8 ns RDFMD (R1–12) simulations.

Figure 5

Representative conformations of the catalytic loop from the clustering of snapshots generated from 4.8 ns of RDFMD and 40 ns of MD simulation. Numbers in brackets depict the % of the total number of snapshots in each cluster. Mn²⁺ is shown in green van der Waals and NADPH cofactor in tan licorice representation. The catalytic loop residues are highlighted in blue with loop residues His200 and Trp203 in red.

Figure 6

Sampling of clusters over 40 ns MD (MD1 and MD2) and 4.4 ns RDFMD (R1–11) simulations of DXR–FMN.

Figure 7

Representative conformations of the catalytic loop from the clustering of snapshots generated from 4.4 ns of RDFMD and 40 ns of MD simulation. Numbers in brackets depict the % of the total number of snapshots in each cluster. Mn²⁺ is shown in green van der Waals and NADPH (tan) and fosmidomcyin in licorice representation. The catalytic loop residues are highlighted in blue with loop residues His200 and Trp203 in red.

Figure 8

RMSD of Cα atoms of the catalytic loop against the crystal structure (PDB ID: 2JCZ). Top two lines in each plot show the RMSD against the open loop (monomer B of 2JCZ) and bottom two lines show the RMSD against the closed loop (monomer A of 2JCZ) monitored over MD1 (red) and MD2 (black) simulations of (A) DXR–MN (B) DXR–FMN.

Figure 9

RMSD of catalytic loop residues measured against crystal structure of open loop (bold) and closed loop (dotted) over the 12 and 11 RDMFD simulations of (A) DXR–MN (B) DXR–FMN respectively.

Figure 10

Loop conformations of three opening events observed in RDFMD simulations of DXR–MN (see numbered labels in Figure 9A). Coloring of loop structures (corresponding label in Figure 9A shown in brackets): red (1), orange (2), yellow (3). Open and closed loop conformations of the starting crystal structure (PDB ID: 2JCZ) are shown in grey (opaque).

Figure 11

Representative inner-product matrix of the first 10 eigenvectors for two RDFMD simulations of DXR–MN that observe opening of the catalytic loop. Similarity between eigenvectors demonstrated by shading. High to low similarity represented by dark to light shading respectively.

Figure 12

View from the top of monomer A of MtDXR. The direction and length of the cone pointing from each of the Cα of the average DXR structure show the direction and magnitude of motion captured by the first eigenvector. N-terminal highlighted in red, C-terminal in green and catalytic domain in orange with the extended loop and loop residues highlighted in yellow and purple respectively.