Reorienting Mechanism of Harderoheme in Coproheme Decarboxylase-A Computational Study

Abstract

Coproheme decarboxylase (ChdC) is an important enzyme in the coproporphyrin-dependent pathway (CPD) of Gram-positive bacteria that decarboxylates coproheme on two propionates at position 2 and position 4 sequentially to generate heme b by using H₂O₂ as an oxidant. This work focused on the ChdC from Geobacillus stearothermophilus (GsChdC) to elucidate the mechanism of its sequential two-step decarboxylation of coproheme. The models of GsChdC in a complex with substrate and reaction intermediate were built to investigate the reorienting mechanism of harderoheme. Targeted molecular dynamics simulations on these models validated that harderoheme is able to rotate in the active site of GsChdC with a 19.06-kcal·mol^-1 energy barrier after the first step of decarboxylation to bring the propionate at position 4 in proximity of Tyr145 to continue the second decarboxylation step. The harderoheme rotation mechanism is confirmed to be much easier than the release-rebinding mechanism. In the active site of GsChdC, Trp157 and Trp198 comprise a "gate" construction to regulate the clockwise rotation of the harderoheme. Lys149 plays a critical role in the rotation mechanism, which not only keeps the Trp157-Trp198 "gate" from being closed but also guides the propionate at position 4 through the gap between Trp157 and Trp198 through a salt bridge interaction.

Keywords: coproheme decarboxylase; rotation; targeted molecular dynamics simulation; two-step decarboxylation.

Figure 1

Coproheme–GsChdC complex highlighting the relevant binding orientations for the substrate. (a) Cartoon representation of pentamer assemblies of representative crystal structures of the coproheme–GsChdC complex: different chains of GsChdC are shown in green for chain A, cyan for chain B, magenta for chain C, yellow for chain D and wheat for chain E, respectively. The coproheme cofactor is depicted as orange sticks. (b) The chemical structure of coproheme (left) and its schematic representation (right). (c) Close-up view of the active site structures binding with a coproheme, highlighting the positions of the side chains for the neighboring Tyr145, Arg131, Lys149, Trp157, His171 and Trp198 in cyan sticks and coproheme in orange sticks.

Figure 2

The complete catalytic process of coproheme decarboxylation of GsChdC, highlighting different relevant binding orientations of coproheme (blue), harderoheme (green) and heme b (yellow) in GsChdC with respect to the catalytic decarboxylation site Tyr145. The color of Tyr145 is switched to orange when it is ready for decarboxylation. The complete catalytic process starts with a binding process of coproheme. The propionate at position 2 (p2) is captured by Tyr145 with a hydrogen bond interaction, thereby initiating its first-step decarboxylation and the formation of vinyl (v2). The produced harderoheme rotates around the Fe–N_ε axis for about 90 degrees clockwise (pose 90) after losing the hydrogen bond interaction between p2 and Tyr145. The propionate at position 4 (p4) is captured by Tyr145 with a hydrogen bond interaction again, which attacks p4 for the second step decarboxylation that forms heme b. Finally, heme b is released and delivered to the cell environment.

Figure 3

Root mean square deviation (RMSD) values of heavy atoms with respect to the initial structure of the monomer model systems, respectively: (a) FM1, (b) FM2, (c) FM3, (d) FM4 and (e) FM5.

Figure 4

Representative structure for FM3 from the MD ensemble. The different secondary structures of the monomer model are marked as cyan for α-helix and magenta for β-sheet.

Figure 5

Time-dependent results from four individual targeted molecular dynamics (TMD) simulations of the harderoheme clockwise rotation process using different harmonic force constants of 0.01, 0.10, 0.50 and 3.00 kcal/(mol·Å²), respectively. (a) RMSD values of all backbone atoms. (b) The evolution of the dihedral angle of C_α(sub-p4)-Fe_(sub)-N_δ(His171)-O_η(Tyr145).

Figure 6

The time-dependent evolution of the harderoheme clockwise rotation of WT and the Lys149Ala mutant. (a,b) MM-PBSA enthalpy diagram of WT and Lys149Ala, respectively. (c) Distances between ammonium N of Lys149 and each of the two carboxylate O of p4 of WT in black and red, respectively, and distances between the C_δ of p4 and the mass centers of two hydrophobic residues Trp157 and Trp198 in blue and magenta, respectively. (d) Distances between the C_δ of p4 and the mass centers of two hydrophobic residues Trp157 and Trp198 in black and red, respectively. (e,f) The dihedral angle C_α(sub-p4)-Fe_(sub)-N_δ(His171)-O_η(Tyr145) of the harderoheme-rotated angle of WT and Lys149Ala, respectively. (g,h) The dihedral angle C_α-C_β-C_γ-C_δ of the p4 side chain of the harderoheme of WT and Lys149Ala, respectively. (i,j) Distances between the mass centers of Trp157 and Trp198 in black and between Met169 and the Fe atom in red of WT and Lys149Ala, respectively. (k,l) RMSD values of heavy atoms with respect to the initial structures of WT and Lys149Ala, respectively. The different background colors denote the different stages of the substrate rotation process: green for stage C, when the substrate is still in the initial structure stage; blue for stage I, when the substrate p4 propionate group crosses through Trp157 and Trp198 in the wild-type system but momentarily blocked in the Lys149Ala system; magenta for stage II, an intermediate stage with relatively low energy; yellow for stage III, where the salt bridges involving the p4 and p7 propionates are successively interrupted in the wild-type system, while, in Lys149Ala, the harderoheme bends under the action of Met169, and p4 manages to cross through Trp157 and Trp198 at this stage and wheat for stage T, when the substrate has evolved into the target structure.

Figure 7

Representative active site structures at different stages of the harderoheme rotation process: (a) WT, stage I, p4 crossing through the Trp157 and Trp198 residues; (b) Lys149Ala, stage III, p4 crossing through the Trp157 and Trp198 residues; (c) WT, stage III, the salt bridge interruption between p4 and Lys149; (d) Lys149Ala, stage III, the salt bridge interruption between p4 and Arg131 and (e) WT, stage III, the salt bridge interruption between p7 and Arg131.

Figure 8

The structures after MD simulations of (a) wild-type GsChdC of harderoheme-pose-0, (b) Lys149Ala of harderoheme-pose-0 and (c) wild-type GsChdC of harderoheme-pose-90. Key distances among the Trp-Lys-Trp portal and the substrate are shown.

Figure 9

MM-PBSA energy decompositions of the barriers into residues for different stages revealed by the TMD simulations: (a) wild-type GsChdC, stage I; (b) wild-type of GsChdC, stage III and (c) Lys149Ala GsChdC, stage III.

Figure 10

Trp–Lys–Trp portal of (a) LmChdC and (b) CdChdC.