The Essential Role of Water Molecules in the Reaction Mechanism of Protein O‐Fucosyltransferase 2

Protein O‐fucosyltransferase 2 (PoFUT2) is an inverting glycosyltransferase (GT) that fucosylates thrombospondin repeats (TSRs) from group 1 and 2. PoFUT2 recognizes a large and diverse number of TSRs through a dynamic network of water‐mediated interactions. By X‐ray structural studies of C. elegans PoFUT2 complexed to a TSR of group 2, we demonstrate that this GT recognizes similarly the 3D structure of TSRs from both groups 1 and 2. Its active site is highly exposed to the solvent, suggesting that water molecules might also play an essential role in the fucosylation mechanism. We applied QM/MM methods using human PoFUT2 as a model, and found that HsPoFUT2 follows a classical SN2 reaction mechanism in which water molecules contribute to a great extent in facilitating the release of the leaving pyrophosphate unit, causing the H transfer from the acceptor nucleophile (Thr/Ser) to the catalytic base, which is the last event in the reaction. This demonstrates the importance of water molecules not only in recognition of the ligands but also in catalysis. PoFUT2 is an inverting glycosyltransferase that fucosylates TSR repeats of group 1 and 2. By applying computational methodologies, it has been found that PoFUT2 follows a pure SN2 mechanism in which water molecules play an essential role in the catalysis. Recognition of the TSR repeats by direct and water‐mediated interactions, together with the role of water molecules around the catalytic center, constitute the driving force of the process.