Revealing the Catalytic Strategy of FTO

Abstract

The fat-mass and obesity-associated protein (FTO) is a Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase of the AlkB family and is linked with obesity and cancer. The enzyme is identified as single-stranded DNA/RNA demethylase with N6-methyladenine (m⁶A) modification in RNA as its most favorable substrate. Herein we used Molecular Dynamics (MD), metadynamics (MetD), and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) calculations to reveal the catalytic mechanism of FTO with pentanucleotide-ssRNA(m⁶A) substrate and elucidate the effects of clinically significant mutations R316Q and S319F. The calculations explored the catalytic mechanism of the O₂ activation and substrate oxidation in the WT FTO, revealing that different networks of residues stabilize the TSs of the different reaction steps. The mutations influence the interactions in the jelly-roll motif and loops in FTO and in particular, S319F strongly affects the substrate binding. The R316Q mutant slows down the O₂ activation and HAT rates in agreement with experimental studies.

Keywords: Catalysis; FTO; Molecular dynamics; Non-heme enzyme; Quantum Mechanics/ Molecular Mechanics (QM/MM); conformational study; mutations.

Figure 1.

Secondary structure of FTO depicting the N-and C-terminal domains (blue and brown), DSBH (cyan), L1 loop (magenta), and the surface loop (yellow). The magnified image in the circle (left) shows the enzyme catalytic site.

Figure 2.

Location of the mutated residues in FTO- R316 (magneta) and S319 (yellow).

Figure 3.

MM MetD of substrate binding to FTO. A) Free energy profile for the binding of m⁶A substrate to the Fe center, B-D) Geometries of the unbound (U), TS and the bound (B) states, respectively, during the substrate binding.

Figure 4.

Energy profiles for O₂ activation by the WT (brown) and mutants R316Q (green), and S319F (blue) with the lowest activation barriers. The relative B2+ZPE is represented in kcal/mol.

Figure 5.

Catalytic site stabilizing residues in the wild-type (WT) ferryl complex (blue). The active site is represented in ball and stick model (yellow).

Figure 6.

Stationary point geometries for the substrate oxidation including the hydrogen atom transfer (HAT) and rebound hydroxylation. The distances (Å) and spin densities are shown in brown and yellow, respectively.

Figure 7.

QM/MM relative energies for the substrate oxidation by WT FTO. A) Energy profile of the reaction path for the substrate hydroxylation. The relative single point (B2) energies are represented in kcal/mol at B3LYP/def2-TZVP (blue) and B2 with ZPE (black). B) The HAT energy barriers for the five studied MD snapshots. Snapshot number 1 was used for further analysis.

Figure 8.

The σ- and π-channels observed for the HAT in high spin (S=2) ferryl complexes. A) σ-channel B) π-channel.

Figure 9.

Schematic molecular orbital representation of transition states involved in the substrate oxidation. A) HAT B) Rebound hydroxylation.

Figure 10.

Energetics for the HAT pathway by WT (brown) and mutant R316Q (green). The relative B2+ZPE is represented in kcal/mol.

Figure 11.

Energetics and stationary point geometries for the second oxidation by WT FTO. The relative single point (B2) energies are represented in kcal/mol at B3LYP/def2-TZVP (red) and B2 with ZPE (black).

Figure 12.

The QM region of the FTO-2OG-ssRNA(m⁶A)Fe(III)-O-O^.- complex used for the QM/MM calculations.

Scheme 1.

Catalytic cycle for the demethylation of m⁶A by FTO.