Revealing the catalytic strategy of FTO

The fat-mass and obesity-associated protein (FTO) is an Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase of the AlkB family and is linked with obesity and cancer. The enzyme is identified as a single-stranded (ss)DNA/RNA demethylase with N6-methyladenine (m6A) modification in RNA as its most favorable substrate. Here 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 wild-type (WT) FTO, revealing that different networks of residues stabilize the transition states 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 hydrogen atom transfer (HAT) rates, in agreement with experimental studies.