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.
Highlights
O-glycosylation is a post-translational protein modification essential to life
The formation of the glycosidic linkages in glycans is catalyzed by glycosyltransferases (GTs), highly specific enzymes that utilize an activated donor sugar substrate that contains a substituted phosphate leaving group.[3]
protein O-fucosyltransferase 1 (POFUT1) is interesting because of its involvement in the Notch signaling pathway (NSP), an essential cell−cell communication pathway conserved in all multicellular animals.[8,9]
Summary
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). One the strengths of metadynamics is that it does not rely on an initial guess of the reaction pathway, and as such the method has been recently used to discover catalytic mechanisms of several glycoside hydrolases (GHs)[21−25] and GTs.[26−29] Our simulations show that the Asn[51] residue plays an essential catalytic role by mediating proton transfer from the Thr hydroxyl group to the leaving acceptor phosphate.
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