Substrate-Guided Front-Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N-Acetylgalactosaminyltransferase 2

The retaining glycosyltransferase GalNAc‐T2 is a member of a large family of human polypeptide GalNAc‐transferases that is responsible for the post‐translational modification of many cell‐surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum‐mechanics/molecular‐mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic‐electronic level of detail. Our study provides a detailed structural rationale for an ordered bi–bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front‐face SNi‐type reaction in which the substrate N‐acetyl sugar substituent coordinates efficient glycosyl transfer. The magic of many moments: Structural snapshots of GalNAc‐T2 complexes during the catalytic cycle were combined with quantum‐mechanics/molecular‐mechanics metadynamics to reveal an ordered bi–bi kinetic mechanism. Critical aspects of substrate recognition were identified that dictate the specificity for acceptor Thr versus Ser residues and enforce a front‐face reaction in which the substrate N‐acetyl sugar coordinates glycosyl transfer.