How to Tell an N from an O: Controlling the Chemoselectivity of Methyltransferases

<i>S</i>-Adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) are important enzymes in numerous biological pathways. They share a common S _<i>N</i> 2 mechanism but act on different nucleophilic substrates in vivo. Therefore, MTs have a specific chemoselectivity to transfer CH₃ onto the correct atom type and substrate. Caffeate <i>O</i>-MT from <i>Prunus persica</i> (<i>Pp</i>CaOMT) and anthranilate <i>N</i>-MT from <i>Ruta graveolens</i> (<i>Rg</i>ANMT) share a high similarity regarding their amino acid sequence (>74%). Nevertheless, the physiological substrates (caffeate vs anthranilate) and attacking nucleophiles (hydroxyl vs amino group) are strikingly different. We demonstrate that the differing chemoselectivity is governed by different conformational states of the two enzymes. <i>O</i>-Methylation catalyzed by CaOMTs requires a "closed" conformation, whereas ANMTs perform <i>N</i>-methylation in an "open" state. We rationally designed seven variants for both <i>Pp</i>CaOMT and <i>Rg</i>ANMT, which changed their original nucleophile preference to different extents, up to a full inversion. Interestingly, the generated <i>O</i>-selective ANMT variant catalyzes <i>O</i>-methylation considerably faster than wildtype CaOMT. Molecular dynamics (MD) simulations and hydrogen/deuterium exchange mass spectrometry (HDX-MS) experiments showed that the mutations induced changes in the conformational dynamics of the enzyme variants and by modulating the open/closed transitions impact the corresponding chemoselectivity. Our data show that the selectivity of the methyl transfer reaction is not solely governed by the key residues directly involved in the methyl transfer but is rather synergistically modulated by the conformational dynamics of the enzyme and reaction conditions.