Mechanistic Studies of 1-Deoxy-D-Xylulose-5-Phosphate Synthase from Deinococcus radiodurans

Abstract

The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is the sole source of these terpenoids for the production of isoprenoids in the apicomplexan parasites, in many eubacteria, and in plants. The absence of this pathway in higher organisms has opened a new platform for the development of novel antibiotics and anti-malarials. The enzyme catalyzing the first step of the NMVA pathway is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). DXPS catalyzes the thiamine pyrophosphate- and Mg (II)-dependent conjugation of pyruvate and D-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate and CO₂. The kinetic mechanism of DXPS from Deinococcus radiodurans most consistent with our data is random sequential as shown using a combination of kinetic analysis and product and dead-end inhibition studies. The role of active site amino acids, identified by sequence alignment to other DXPS proteins, was probed by constructing and analyzing the catalytic efficacy of a set of targeted site-directed mutants.

Keywords: Dimethylallyl pyrophosphate; Isopentenyl pyrophosphate; Non-mevalonate; Site-directed mutagenesis; TPP-dependent; α-Carbanion/Enamine intermediate.

Figure 1

The reaction catalyzed by DXPS (R1=4′-amino-2-methyl-5-pyrimdyl and R2=β-hydroxyethyldiphosphate).

Figure 2

Schematic of D. radiodurans DXPS depicting the bound TPP. This figure was created using Maestro 9.3.023 from Schrödinger suite 2012 and PDB 2o1x. Domain I of D. radiodurans DXPS is shown as purple ribbon, domain II of D. radiodurans DXPS is shown as green ribbon as ribbon, TPP is shown as tube with aquamarine carbon atoms, and the Mg (II) ion is shown as a pink sphere.

Figure 3

Domain I of D. radiodurans DXPS including the bound TPP molecule. Domain I mutant enzymes (H82A, N181A, N183, and H304A) with the dashed lines representing hydrogen bonds and key residue highlighted in deep blue. The models in the column on the left show the wild-type enzyme while the mutant enzymes are shown in the models in the column on the right. These models do not represent all possible differences in hydrogen bonding due to the lack of substrates in the active but, no crystal structures of DXS currently exist containing the bound. The models were prepared utilizing PyMOL.

Figure 4

Domain II of D. radiodurans DXPS including the bound TPP molecule. Domain II mutant enzymes (Y395A, Y395F, R423A, R423K, D430A, and the H434A) with the dashed lines representing hydrogen bonds and key residue highlighted in deep blue. The left column on the far left is a model for the wild-type enzyme while the models in the right column images illustrate the mutant enzymes. The top 2 rows are the three models that correlate with the Y395 and R423 mutants, respectively. The models were prepared utilizing PyMOL.

Figure 5

Double reciprocal analysis of initial velocities of pyruvate and GAP at different fixed concentrations of the other substrate. Right panel: kinetic analysis was performed using sub-saturating concentrations of GAP: 0.01 mM (●), 0.014 mM (○), 0.02 mM (▼), and 0.03 mM (△). Left panel: kinetic analysis was performed using subsaturating concentration of pyruvate: 0.02 mM (●), 0.03 mM (○), 0.04 mM (▼), and 0.06 mM (△).

Figure 6

Double reciprocal analyses of initial velocities of DXPS inhibition by DXP at different initial concentrations of pyruvate (right panel) and GAP (left panel). Kinetic analyses were performed using the following concentrations of DXP: 0 mM (●), 0.2 mM (○), 0.3 mM (▼) and 0.4 mM (▽).

Figure 7

Double reciprocal analyses of initial velocities of DXPS inhibition by F-Pyr at different fixed concentration of pyruvate (right panel) and GAP (left panel). Kinetic analysis was performed using the following concentrations of F-Pyr: 0 μM (●), 25 μM (○), 50 μM (▼), 75 μM (▽), and 100 μM (■).

Figure 8

The random sequential pathway proposed for the reaction catalyzed by D. radiodurans DXPS. Pyr* is the C2-α-carbanion/enamine intermediate as shown in Figure 1.