Asparagine Tautomerization in Glycosyltransferase Catalysis. The Molecular Mechanism of Protein O-Fucosyltransferase 1

Abstract

O -glycosylation is a post-translational protein modification essential to life. One of the enzymes involved in this process is protein O -fucosyltransferase 1 (POFUT1), which fucosylates threonine or serine residues within a specific sequence context of epidermal growth factor-like domains (EGF-LD). Unlike most inverting glycosyltransferases, POFUT1 lacks a basic residue in the active site that could act as a catalytic base to deprotonate the Thr/Ser residue of the EGF-LD acceptor during the chemical reaction. Using quantum mechanics/molecular mechanics (QM/MM) methods on recent crystal structures, as well as mutagenesis experiments, we uncover the enzyme catalytic mechanism, revealing that it involves proton shuttling through an active site asparagine, conserved among species, which undergoes tautomerization. This mechanism is consistent with experimental kinetic analysis of Caenorhabditis elegans POFUT1 Asn43 mutants, which ablate enzyme activity even if mutated to Asp, the canonical catalytic base in inverting glycosyltransferases. These results will aid inhibitor development for Notch-associated O -glycosylation disorders.

Enhanced-Sampling Simulations for the Estimation of Ligand Binding Kinetics: Current Status and Perspective

“…These approaches can tackle important biomedicine problems such as the study of transition-metal-based drugs binding to proteins ( Calandrini et al, 2015 ) or the description of enzymatic reactions. ( Carloni et al, 2002 ; Liao and Thiel, 2013 ; Roston et al, 2016 ; Caldararu et al, 2018 ; Kulik, 2018 ; Piniello et al, 2021 ) However, these simulations are orders of magnitude more expensive than any of the potentials described so far, and hence achieving high statistical accuracy with such an approach is obviously extremely challenging.…”

Computer Simulation to Rationalize “Rational” Engineering of Glycoside Hydrolases and Glycosyltransferases

“…One mechanistic exception are the family GT6 enzymes, such as mammalian α-1,3-galactosyltransferase (α3GalT) and blood-group A and B α-1,3-glycosyltransferases (GTA/GTB), which can catalyze the formation of the glycosidic bond via a double-displacement mechanism (Scheme b). , Unlike other GT families described so far, the active site of GT6 enzymes contains a catalytic nucleophile (glutamate) that is able to attack the anomeric carbon of the donor sugar; thus, the reaction can be completed in two steps, similarly to retaining GHs. Concerning inverting GTs, they are expected to follow a one-step S N 2 reaction (Scheme c), similar to inverting GHs, such as the recently characterized N -acetylglucosaminyltransferase V (GnT-V; family GT18), , but unconventional mechanisms such as asparagine tautomerization (in protein O -fucosyltransferase 1, POFUT1; family GT65) or substrate-assisted catalysis (in O -GlcNAc glycosyltransferase, OGT; family GT41) have also been proposed.…”

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

“…For PoFUT1, a distinct mechanism was found in which this enzyme still followed an S N 2 mechanism, but an Asn residue rather than a basic residue deprotonated the hydroxyl group of the acceptor nucleophile. The Asn residue bridged the nucleophile residue with the negatively charged β‐phosphate, shuttling a proton between the two groups [8] …”