N- and S-Oxidation Model of the Flavin-containing MonoOxygenases Peter J. Walton, Mario Ӧeren, Peter Hunt, Matthew Segall Optibrium, StarDrop, Auto-Modeller, Card View, Glowing Molecule and Augmented Chemistry are trademarks of Optibrium Ltd.
Background Flavin-containing MonoOxygenase (FMO) 5 active isoforms (FMO1 5) FMO3 major isoform found in adult liver Flavin Moiety Enzyme class found in multiple tissues Phase I enzyme involved in compound metabolism Metabolites commonly subject to Phase II metabolism PDB ID: 2XVE 2
Role of Flavin-containing Monooxygenases Works in conjunction with Cytochrome P450 in Phase I metabolism FMO3 polymorphism responsible for trimethylaminuria Decreased rate of metabolism leads to increased drug exposure Variety of reaction types possible: N/S-Oxidation 99% cases Demethylation, Bayer-Villiger Oxidation, Desulfuration Trimethylamine Chlorpromazine Voriconazole Fenthion Tozasertib 3
N/S Oxidation N/S-Oxidation involves transfer of Oxygen from peroxide of FAD OOH Debate in the literature regarding mechanism: Radical S N 2 Our focus: Better understanding of the mechanism substrate oxidation by FAD peroxide. 4
Model System
Simplifying the System Full FAD too large for practical calculations Tail buried in groove and does not interact with substrate Compared bond stretching of the full FAD OOH vs the simplified analogue Behaviour of simplified system at peroxide site analogous to larger molecule DFT Energy (kj mol -1 ) 160 140 120 100 80 60 40 20 0 DFT Energy vs r O-O for FAD and Simplified FAD 1.45 1.5 1.55 1.6 1.65 1.7 1.75 1.8 1.85 1.9 1.95 2 O-O Bond Length (Å) FAD Simplified FAD B3LYP/def2-SVP 6
Finding the Transition State We searched for both S N 2 and radical transition state Despite multiple attempts we were unable to find a transition state for a radical mechanism Transition state found for closed-shell system, S N 2 mechanism indicative of B3LYP/def2-SVP Bach R. D., J. Phys. Chem. A 2011, 155, 40, 11087-11100 7
Analysis of the S N 2 Mechanism
Thermodynamic Feasibility 900 N-O bond length (Å) 2.4 2.2 2.0 1.8 1.6 1.4 60 kj mol -1 Interaction Energy (kj mol -1 ) 800 700 600 500 400 300 200 100 O-O N-O 233 kj mol -1 0 1.4 1.6 1.8 2.0 2.2 2.4 O-O bond Length (Å) 73 kj mol -1 B3LYP/def2-SVP QTAIM 9
Electron Density Reactant e Bohr -3 B3LYP/def2-SVP 10
Electron Density Pre-transition State e Bohr -3 B3LYP/def2-SVP 11
Electron Density Transition State e Bohr -3 B3LYP/def2-SVP 12
Electron Density Post-transition State e Bohr -3 B3LYP/def2-SVP 13
Electron Density Comparison of reactant and post-transition state structures B3LYP/def2-SVP 14
Properties Derived from Electron Density
Charge Analysis -0.1-0.1-0.1 +0. 1-0.3-0.3-0.1 Isolated FAD OOH Transition State Hirshfeld Charge 16
Charge Analysis +0. 1-0.3-0.3 - -0.4-0.1-0.1 Transition State FAD O- Anion Hirshfeld Charge 17
Charge Analysis +0. 1-0.3-0.3. -0.1-0.1-0.1 Transition State FAD O. Radical Charges seen in transition state more consistent with S N 2 mechanism Hirshfeld Charge 18
Orbital Investigation Orbital shape and orientation conducive to successful interaction Lone pair of trimethylamine (TMA) large component of HOMO O O σ* component of the LUMO of FAD OOH accessible HOMO of TMA LUMO of FAD OOH B3LYP/def2-SVP 19
Full S N 2 Mechanism
Constrained Optimisation Fixed N O bond length = 5.00 Å Pre-optimised Structure Post-optimised Structure B3LYP/def2-SVP 21
Constrained Optimisation Fixed N O bond length = 1.85 Å Pre-optimised Structure Post-optimised Structure B3LYP/def2-SVP 22
Constrained Optimisation Fixed N O bond length = 1.70 Å Pre-optimised Structure Post-optimised Structure B3LYP/def2-SVP 23
Full Concerted Mechanism Hydrogen transfer during optimisation shows no secondary local minima B3LYP/def2-SVP 24
Application to Other Molecules 121 kj mol -1 62 kj mol -1 Observed Non-Observed 156 kj mol -1 20 kj mol -1 70 kj mol -1 57 kj mol -1 108 kj mol-1 28 kj mol -1 177 kj mol -1 133 kj mol -1 80 kj mol -1 69 kj mol -1 90 kj mol -1 B3LYP/def2-SVP 25
Summary Identified transition state for closed-shell (S N 2) mechanism Extensive tests further support S N 2 mechanism Modelling of reaction coordinate provides a greater understanding of the N/S-Oxidation process Further tests indicate that this mechanism is applicable to larger substrates This enables further efforts to predict FMO metabolism 26
Thank You! Mario Ӧeren Peter Hunt Matthew Segall Jonathan Hirst 27