Revealing the Catalytic Strategy of FTO

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.