Theoretical Study On The Mechanism Of Caffeine Metabolism By Cytochrome P450 And Flavin Monooxygenase FMO

Caffeine, an important purine alkaloid, is always added to food, beverage and drug and therefore is closely related to human health. Bioconversion of caffeine into its metabolites is an enzymatic process occurring in liver. Cytochrome P450(CYP) and flavin-containing monooxygenase(FMO) presented in the liver microsomes are the main enzymes catalyzing the metabolism of caffeine. It is of significant importance to explore the enzymatic metabolic mechanisms of caffeine for the development of bio-decaffeination techniques.The major metabolic pathways of caffeine include N-demthylation and C8-oxdation. The overall N-demethylation process of caffeine proceeds in two discrete stages: N-methyl hydroxylation to form the carbinolamine followed by carbinolamine decomposition via proton transfer to yield the demethylated metabolite. Theobromine(TB), paraxanthine(PX) and theophylline(TP) are formed by N-demethylation at N1-, N3- and N7-site, respectively. Besides, caffeine can also be oxidized at C8-site, yielding 1,3,7-trimethyluric acid(TMU). C8-oxidation is also stepwise, including the nucleophilic attack of oxygen atom at C8 atom to generate 8-oxo-caffeine and subsequent intramolecular proton transfer of 8-oxo-caffeine. Then, the metabolic mechanisms of caffeine catalyzed by P450 and FMO were systematically pursued in this study based on the density functional theory.First, the metabolic mechanisms of caffeine catalyzed by CYP1A2, one of the important P450 isoenzymes, were explored in detail in this study using the spin-unrestricted functional of UB3 LYP method. Four metabolic pathways of caffeine were discussed, namely N1-, N3- and N7-demethylations(paths I-III) and C8-oxidation(path IV). The calculated results show that the rate-limiting step of N-demethylation involves the N-methyl hydroxylation, which proceeds through a hydrogen atom transfer(HAT) mechanism. The subsequent carbinolamine decomposition prefers to the adjacent heteroatom-assisted proton transfer mechanism in a non-enzymatic environment. The rate-limiting step of C8-oxidation involves the nucleophilic attack of the active Cpd I’s oxygen at C8 atom. The obtained results suggest that all reaction pathways take place in a two-state reactivity mechanism(TSR). Caffeine metabolic performance depended on the multiplicity of Cpd I. On the high-spin quartet state, N3-demethylation(path II) is the optimum metabolic pathway of caffeine due to its lowest activation energy(14.7 kcal mol-1) and paraxanthine is therefore the most energetically feasible metabolite of caffeine catalyzed by CYP1A2. On the low-spin doublet state, however, C8-oxidation(path IV) has the lowest activation energy(16.6 kcal mol-1) and is the optimum metabolic pathway. Thus, 1,3,7-trimethyluric acid is the optimum metabolite of caffeine.And then, dispersion corrected hybrid functional of B3LYP-D3 was adopted to discover the metabolic mechanisms of caffeine catalyzed by the active FLHOOH of FMO. The metabolic processes of caffeine catalyzed by FMO are similar to those by P450, including N1-, N3- and N7-demethylation and C8-oxidation. The calculated results imply that the rate-limiting step of N-demethylation is the N-methyl hydroxylation, which is a radical reaction mechanism involving the homolysis of C-H and O-O bonds and an incomplete somersault rearrangement of –OOH of FLHOOH. The rate-limiting step for C8-oxidation still involves the nucleophilic attack of oxygen atom via an oxygen atom transfer mechanism. Both the carbinolamine decomposition and the intramolecular proton transfer of 8-oxo-caffeine are more prone to the adjacent heteroatom-assisted proton transfer. C8-oxidation is the optimal reaction pathway due to its lowest activation energy(25.6 kcal mol-1) and trimethyluric acid is therefore the optimum metabolite of caffeine catalyzed by FMO.Based on the metabolic mechanisms of caffeine by the active FLHOOH of FMO, the effects of key residues Asn91 and Thr92 in FMO active site were further explored based on the rate-determining step of caffeine in help of clear understanding the role of the residue. First, individual influence of residues Asn91 and Thr92 on the caffeine metabolic processes were investigated in turn. And then the cooperation influence of residues Asn91 and Thr92 on the thermodynamics and kinetics property of caffeine metabolic processes was identified. The obtained results show that the presence of single residue Asn91 contributes to the stability of transition states via H-bonding interactions with the OOH group in FLHOOH and caffeine, which results in the decreases of activation energies of 1.7~3.4 kcal mol-1 than direct reaction of caffeine with FLHOOH. Similarly, the presence of single residue Thr92 has H-bonding interactions with the isoalloxazine ring of FLHOOH and caffeine, leading to the decreases of activation energies of 1.4~3.4 kcal mol-1. When both Asn91 and Thr92 were considered, residue Asn91 has H-bonding interaction with OOH group of FLHOOH and caffeine, whereas residue Thr92 has H-bonding interactions with the isoalloxazine ring of FLHOOH at O2 and N3-H sites. The activation energies are reduced by 1.3~4.5 kcal mol-1. Consequently, residues Asn91 and Thr92 are both favorable for the metabolism of caffeine catalyzed by FMO. This work has shed light on the roles of the key residues Asn91 and Thr92 in FMO on the metabolism of caffeine, which can provide more comprehensive information for the metabolic mechanisms of caffeine catalyzed by FMO.