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. 2023 Mar 23;24(7):6064.
doi: 10.3390/ijms24076064.

Hydroperoxidation of Docosahexaenoic Acid by Human ALOX12 and pigALOX15-mini-LOX

Miquel Canyelles-Niño  1   2 , Àngels González-Lafont  1   3 , José M Lluch  1   3
Affiliations

Hydroperoxidation of Docosahexaenoic Acid by Human ALOX12 and pigALOX15-mini-LOX

Miquel Canyelles-Niño et al. Int J Mol Sci. .

Abstract

Human lipoxygenase 12 (hALOX12) catalyzes the conversion of docosahexaenoic acid (DHA) into mainly 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-H(p)DHA). This hydroperoxidation reaction is followed by an epoxidation and hydrolysis process that finally leads to maresin 1 (MaR1), a potent bioactive specialized pro-resolving mediator (SPM) in chronic inflammation resolution. By combining docking, molecular dynamics simulations, and quantum mechanics/molecular mechanics calculations, we have computed the potential energy profile of DHA hydroperoxidation in the active site of hALOX12. Our results describe the structural evolution of the molecular system at each step of this catalytic reaction pathway. Noteworthy, the required stereospecificity of the reaction leading to MaR1 is explained by the configurations adopted by DHA bound to hALOX12, along with the stereochemistry of the pentadienyl radical formed after the first step of the mechanism. In pig lipoxygenase 15 (pigALOX15-mini-LOX), our calculations suggest that 14S-H(p)DHA can be formed, but with a stereochemistry that is inadequate for MaR1 biosynthesis.

Keywords: QM/MM calculations; enzyme catalysis; human platelet ALOX12; hydroperoxidation mechanism; molecular dynamics simulations.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Protein and substrate backbone RMSDs versus time for the MD replica 1 and replica 2 in hALOX12.
Figure 2
Figure 2
PLAT domain of hALOX12 in the closed (in green) and in the open (in orange) conformation. The C-terminal domain is shown in grey.
Figure 3
Figure 3
DHA binding mode in the closed (in green) and the open (in red) conformations of hALOX12. The interaction between Arg402 and the carboxylate group of DHA is shown as well as the rotation of Phe414.
Figure 4
Figure 4
Distances of a carboxylate’s oxygen atom of DHA to the closest hydrogen atom in Arg402 and Gln406 versus time for the MD replica 1 and replica 2 in hALOX12.
Figure 5
Figure 5
Distance between the oxygen atom in Gln406 and the closest hydrogen atom in Arg402 versus time for the MD replica 1 in hALOX12.
Figure 6
Figure 6
Distances between Phe174 side chain and the closest atom of the Δ4 and Δ7 double bonds versus time for the MD replica 1 and replica 2 in hALOX12.
Figure 7
Figure 7
Main stacking interactions between hALOX12 residues and DHA. The protein residues and DHA are shown in green for the closed conformation, and in orange and red, respectively, for the open conformation of the MD replica 1.
Figure 8
Figure 8
Main interactions between the terminal methyl of DHA and residues of the bottom of hALOX12′s cavity for the close (upper, blue) and open (lower, orange) conformations.
Figure 9
Figure 9
Evolution with time of the H12-OH−, H9-OH−, and H15-OH− distances along the MD replica 1 and replica 2 in hALOX12.
Figure 10
Figure 10
Evolution with time of the angle between planes formed by C7C8C9 and C9C10C11 (in red) and by C10C11C12 and C12C13C14 (in blue) along the MD replica 1 in hALOX12.
Figure 11
Figure 11
Scheme of the DHA hydroperoxidation mechanism. PCET stands for proton coupled electron transfer.
Figure 12
Figure 12
Images of the optimized reactant (a), transition state structure (b), and product (c) of the H12proS abstraction initiated from snapshot 8721 in hALOX12. The distance between H12proS and the oxygen atom in OH− is shown for the optimized reactant (a). At the transition state structure, the distances between the shifting hydrogen with respect to C12 (donor atom) and the oxygen atom in OH− (acceptor atom) are depicted (b). The distance between the H atom in the nascent water molecule and C12 is plotted at the optimized product (c).
Figure 13
Figure 13
Images of the optimized reactant (a), transition state structure (b), and product (c) of the O2 addition at C14 (frame 8721) in hALOX12. The distances between C14 and the attacking oxygen atom are given for the reactant and transition state structures.
Figure 14
Figure 14
Images of the optimized reactant (a), transition state structure (b), and product (c) of the carbon chain rotation of the peroxyl radical (frame 8721) in hALOX12.
Figure 15
Figure 15
Images of the transition state structure (TS1) (a), intermediate (INT1) (b), transition state structure (TS2) (c), and the final 14S-H(p)DHA product (d) (frame 8721) in hALOX12. The distances between the outer oxygen of the peroxo group and the water hydrogen atom are given for TS1 and INT1. The distances corresponding to the shifting hydrogen atom are indicated for TS2 and the final 14S-H(p)DHA product.
Figure 15
Figure 15
Images of the transition state structure (TS1) (a), intermediate (INT1) (b), transition state structure (TS2) (c), and the final 14S-H(p)DHA product (d) (frame 8721) in hALOX12. The distances between the outer oxygen of the peroxo group and the water hydrogen atom are given for TS1 and INT1. The distances corresponding to the shifting hydrogen atom are indicated for TS2 and the final 14S-H(p)DHA product.
Figure 16
Figure 16
Overall energy scheme of the H12proS-abstraction, oxygen addition, carbon chain rotation, and retro-hydrogen abstraction steps of the DHA hydroperoxidation mechanism in hALOX12. All energies are in kcal/mol. The zero of energies corresponds to the reactant of the H12proS abstraction step bound to the solvated enzyme plus an oxygen molecule within the water box whose position has been optimized at 12.1 Å from the substrate’s C14.
Figure 17
Figure 17
Distances of a carboxylate’s oxygen atom of DHA to the closest hydrogen atom in Arg403 versus time for the MD replica 1 and replica 2 in pigALOX15-mini-LOX.
Figure 18
Figure 18
Distances between Phe175 sidechain and the closest atom of the Δ4 and Δ7 double bonds versus time for the MD replica 1 and for replica 2 in pigALOX15-mini-LOX.
Figure 19
Figure 19
Evolution with time of the H12-OH−, H9-OH−, and H15-OH− distances along the MD replica 1 and replica 2 in pigALOX15-mini-LOX.
Figure 20
Figure 20
Evolution with time of the angle between planes formed by C7C8C9 and C9C10C11 (in red) and by C10C11C12 and C12C13C14 (in blue) along the MD replica 1 in pigALOX15-mini-LOX.
Figure 21
Figure 21
QM/MM partition in the DHA/hALOX12 (a) and DHA/pigALOX15-mini-LOX systems (b).
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