Figure 1.
Structural overview of native and oxidized phenylalanine hydroxylase.
The catalytic domain of native phenylalanine hydroxylase in blue cartoon representation (top left). The biopterin co-factor in the active site is shown as sticks, the catalytic iron as brown sphere. Cysteine residues 203 and 334 are shown as sticks to highlight the artificially introduced oxidation site distant from the catalytic center (red, top right). A zoom at the site of oxidation at the back of the top figures is shown at the bottom. Cys203 and Cys334 in proximity to each other (native state, blue, bottom left) are closed to a disulfide bond (oxidized state, red, bottom right).
Figure 2.
Global dynamic behavior of native and oxidized phenylalanine hydroxylase.
A: RMSD of Cα-atoms over the simulation time of 200 ns standard MD simulations for the two systems PAHnat (blue) and PAHox (red). After an initial phase of loop reorientation for the oxidized system PAHox (around 20 ns simulation time), simulations yield stable trajectories. B: Residue-wise B-factors for the simulated systems highlight residues 130–145 as particularly altered by the introduced cysteine oxidation. Elevated B-factors in this loop region (Tyr138-loop) in PAHox are caused by a reorientation of the loop in an early stage of the simulation. Dynamics of residues around the oxidation sites (Cys-203, Cys334) are similar in both systems.
Figure 3.
Dynamics of the binding site region of native and oxidized phenylalanine hydroxylase.
A: Distance between the catalytic iron center and the Cα-atom of Glu141 included in the flexible Tyr138-loop near the active site. Whereas this distance remains mostly stable in the native state PAHnat over the simulation time (blue), simulation of PAHox shows an initial phase of increased loop flexibility that is followed by decrease of that distance indicating a loop movement towards the active site. This finding is confirmed by calculation of the accessible surface area of the iron center (B). After an initial adaption to the perturbation, PAHox shows a constantly reduced accessibility of the catalytic iron center compared to PAHnat. The overall active site volume (C) is similarly reduced over simulation time in PAHox compared to PAHnat, suggesting a reduced enzyme turnover.
Table 1.
Averages and standard deviations (SD) of accessible surface area of the catalytic iron as well as the total active site volume for the simulations of PAHnat and PAHox.
Figure 4.
Changes in the active site of phenylalanine hydroxylase introduced by side chain oxidation.
Starting coordinates of both simulations (blue cartoon) overlaid with an average structure of the time frames 150–160 ns of standard MD simulation PAHox (red cartoon). The pronounced loop movement towards the catalytic center (iron ion as brown sphere, co-factor as sticks) was measured as distance of the Cα-atom of Glu141 (red and blue sphere respectively) to the catalytic iron (brown). This rearrangement is paralleled by a reorientation of Tyr138 (shown as sticks, left), in turn concertedly blocking access to the active site of PAHox.
Figure 5.
Alteration in local dynamics in phenylalanine hydroxylase upon side chain oxidation.
Residue-wise B-factors for the 2*16 REMD trajectories were calculated for Cα-atoms and compared between simulations of equal temperatures. Rank-based differences were mapped on the protein structure, where blue regions indicate regions more flexible in more of the 16 simulations of PAHnat. Unaffected regions are shown in white, whereas red regions indicate regions showing elevated backbone dynamics in more of the 16 simulations of PAHox. Elevated B-factors in the central helices suggest an allosteric signal transduction over this path from the introduced oxidation site (Cys203, Cys334) to the Tyr138-loop near the catalytic center. The bottom picture shows the same structure rotated by 220° to highlight local effects on the oxidation site in the back of the top picture.