Mechanism of Melatonin Metabolism by CYP1A1: What Determines the Bifurcation Pathways of Hydroxylation versus Deformylation?

Abstract

Melatonin, a widely applied cosmetic active ingredient, has a variety of uses as a skin protector through antioxidant and anti-inflammatory functions as well as giving the body UV-induced defenses and immune system support. In the body, melatonin is synthesized from a tryptophan amino acid in a cascade of reactions, but as melatonin is toxic at high concentrations, it is metabolized in the human skin by the cytochrome P450 enzymes. The P450s are diverse heme-based mono-oxygenases that catalyze oxygen atom-transfer processes that trigger metabolism and detoxification reactions in the body. In the catalytic cycle of the P450s, a short-lived high-valent iron(IV)–oxo heme cation radical is formed that has been proposed to be the active oxidant. How and why it activates melatonin in the human body and what the origin of the product distributions is, are unknown. This encouraged us to do a detailed computational study on a typical human P450 isozyme, namely CYP1A1. We initially did a series of molecular dynamics simulations with substrate docked into several orientations. These simulations reveal a number of stable substrate-bound positions in the active site, which may lead to differences in substrate activation channels. Using tunneling analysis on the full protein structures, we show that two of the four binding conformations lead to open substrate-binding pockets. As a result, in these open pockets, the substrate is not tightly bound and can escape back into the solution. In the closed conformations, in contrast, the substrate is mainly oriented with the methoxy group pointing toward the heme, although under a different angle. We then created large quantum cluster models of the enzyme and focused on the chemical reaction mechanisms for melatonin activation, leading to competitive O-demethylation and C6-aromatic hydroxylation pathways. The calculations show that active site positioning determines the product distributions, but the bond that is activated is not necessarily closest to the heme in the enzyme–substrate complex. As such, the docking and molecular dynamics positioning of the substrate versus oxidant can give misleading predictions on product distributions. In particular, in quantum mechanics cluster model I, we observe that through a tight hydrogen bonding network, a preferential 6-hydroxylation of melatonin is obtained. However, O-demethylation becomes possible in alternative substrate-binding orientations that have the C6-aromatic ring position shielded. Finally, we investigated enzymatic and non-enzymatic O-demethylation processes and show that the hydrogen bonding network in the substrate-binding pocket can assist and perform this step prior to product release from the enzyme.

Biotransformation of Bisphenol by Human Cytochrome P450 2C9 Enzymes: A Density Functional Theory Study

“…Geometrically, the Fe–O distance is short and typically around 1.63 Å for model A and 1.65 Å for the model B structures, which matches previous calculations on P450 CpdI well. − ,,,, The Fe–S bond is rather long, and specifically with the axial protein chain included in model B , it is around 2.609 Å for 2 Re B and 2.640 Å for 4 Re B , while for the truncated model A the distances are slightly shorter. Previous studies using either QM/MM or DFT cluster models gave CpdI Fe–S distances in the range 2.512–2.676 Å; ,,,− hence, our calculated values fit that window nicely. The optimized geometry compares well with the crystal structure coordinates, and an overlay of the two (left-hand side of Figure ) gives a good match.…”

Melatonin Activation by Cytochrome P450 Isozymes: How Does CYP1A2 Compare to CYP1A1?

“…As shown previously [ 65 , 66 , 67 , 68 , 69 ], the O -demethylation starts with the aliphatic hydroxylation of the methoxy group of melatonin and is followed by deformylation through a solvent (and/or proton) assisted step. The latter is expected to happen rapidly in solution and previous calculations reported small barriers for this reaction step [ 65 , 66 , 67 , 68 , 69 ]. Only the aliphatic hydroxylation takes place in the protein; hence, we focus on this step solely.…”

“…Clearly, the substrate positioning is ideal for aromatic hydroxylation of melatonin. Interestingly, the barrier is also notably lower in energy than that found for our CYP1A1 model, where barriers of 10.0 (doublet) and 13.7 (quartet) kcal mol −1 were found [ 65 ]. The first and the last steps in the mechanism release a considerable amount of energy and lead to IM1 and products with high exothermocity.…”

“…Both structures have an imaginary frequency for the C−O stretch vibration and implicate oxygen atom transfer. The values of i240 and i411 cm −1 are typical for aromatic hydroxylation barriers [ 65 , 70 , 71 , 72 , 73 , 74 , 75 , 76 ]. The C−O bond lengths are relatively long, at 2.052 Å in the doublet spin state and 1.887 Å in the quartet spin state.…”

“…Endogenous and exogenous melatonin is degraded to 6-hydroxymelatonin and N -acetylserotonin by aromatic hydroxylation and O -demethylation, respectively [ 60 , 61 , 62 , 63 , 64 ], as shown in Figure 2 . The mechanism of O -demethylation is initiated by a hydrogen atom abstraction from the methoxy group of melatonin to the oxygen atom of Cpd I, which generates a substrate radical side chain and Cpd II [ 65 , 66 , 67 , 68 , 69 ]. The latter, through OH rebound to the radical, gives hydroxylated methoxy group products.…”