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ACS Catal,
2021]
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.
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Angew Chem Int Ed Engl,
2022]
Protein O -fucosyl transferase 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. Here, by X-ray structural studies of C. elegans PoFUT2 complexed to a TSR of group 2, we demonstrate that this GT recognizes similarly both the 3D-structure of TSRs from groups 1 and 2. In addition, 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 the human PoFUT2 as a model, finding that Hs PoFUT2 follows a classical S N 2 reaction mechanism in which waters 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, being this the last event in the reaction. This evidences the importance of water molecules not only in recognition of the ligands but also in catalysis.
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J Mol Graph Model,
2015]
Five sets of 1 GABAC homology models were generated based on X-ray crystal structures of the acetylcholine binding protein (AChBP), the ion channel from Caenorhabditis elegans (GLIC), the ion channel from Erwinia chrysanthemi (ELIC), the homomeric GABAA 3 ion channel, and the homomeric -subunit of glutamate-gated homopentameric chloride channel (GluCl). The GluCl based model was found to the represent the structure of 1 GABAC receptors. The GABA pose docked in the selected best model was confirmed by QM-polarized ligand docking and induced fit docking protocol, and used to study molecular interactions in the 1 GABA binding site. The potential interactions of identified residues are discussed. This study identified several residues with potential ligand interactions located on loops F and G with their side chain oriented toward the binding site such as Ser215 and Gln83. The partial agonists muscimol and imidazole-4-acetic acid (I4AA) were docked into the binding site of the most reliable 'GABA bound' homology model. The potency and efficacy of these partial agonists in activating recombinant 1 receptors were correlated with their docking results. The model predicts that muscimol resembles GABA in the docking pose with similar interactions. However, I4AA has a very different docking pose to GABA and was predicted by the model to form - stacking with aromatic residues in the orthosteric binding site. A set of TPMPA bound 1 homology models based on the GluCl 'apo state' template was built in order to study a competitive antagonist in the 1 orthosteric binding site. The results demonstrated the ability of our model to explain most experimental findings and predict potential roles of residues within the orthosteric binding site.