Substrate-Assisted Catalytic Mechanism of <i>O</i>‑GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding <i>O</i>-linked <i>N</i>-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of <i>O</i>-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer’s disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase <i>O</i>-GlcNAc transferase (uridine diphospho-<i>N</i>-acetylglucosamine:polypeptide β-<i>N</i>-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM­(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme–substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11 326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S_N2-like mechanism, in which a nucleophilic attack by O_Ser, facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1–O_Ser = 1.92 Å and C1–O1 = 3.11 Å. The activation energy for this passage was estimated to be ∼20 kcal mol^–1. These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the <i>N</i>-acetamino group of the donor participates in the catalytic mechanism

Substrate-Assisted Catalytic Mechanism of <i>O</i>‑GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding <i>O</i>-linked <i>N</i>-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of <i>O</i>-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer’s disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase <i>O</i>-GlcNAc transferase (uridine diphospho-<i>N</i>-acetylglucosamine:polypeptide β-<i>N</i>-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM­(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme–substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11 326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S_N2-like mechanism, in which a nucleophilic attack by O_Ser, facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1–O_Ser = 1.92 Å and C1–O1 = 3.11 Å. The activation energy for this passage was estimated to be ∼20 kcal mol^–1. These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the <i>N</i>-acetamino group of the donor participates in the catalytic mechanism

Substrate-Assisted Catalytic Mechanism of <i>O</i>‑GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding <i>O</i>-linked <i>N</i>-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of <i>O</i>-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer’s disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase <i>O</i>-GlcNAc transferase (uridine diphospho-<i>N</i>-acetylglucosamine:polypeptide β-<i>N</i>-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM­(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme–substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11 326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S_N2-like mechanism, in which a nucleophilic attack by O_Ser, facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1–O_Ser = 1.92 Å and C1–O1 = 3.11 Å. The activation energy for this passage was estimated to be ∼20 kcal mol^–1. These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the <i>N</i>-acetamino group of the donor participates in the catalytic mechanism

Substrate-Assisted Catalytic Mechanism of <i>O</i>‑GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding <i>O</i>-linked <i>N</i>-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of <i>O</i>-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer’s disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase <i>O</i>-GlcNAc transferase (uridine diphospho-<i>N</i>-acetylglucosamine:polypeptide β-<i>N</i>-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM­(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme–substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11 326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S_N2-like mechanism, in which a nucleophilic attack by O_Ser, facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1–O_Ser = 1.92 Å and C1–O1 = 3.11 Å. The activation energy for this passage was estimated to be ∼20 kcal mol^–1. These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the <i>N</i>-acetamino group of the donor participates in the catalytic mechanism

Substrate-Assisted Catalytic Mechanism of <i>O</i>‑GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding <i>O</i>-linked <i>N</i>-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of <i>O</i>-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer’s disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase <i>O</i>-GlcNAc transferase (uridine diphospho-<i>N</i>-acetylglucosamine:polypeptide β-<i>N</i>-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM­(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme–substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11 326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S_N2-like mechanism, in which a nucleophilic attack by O_Ser, facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1–O_Ser = 1.92 Å and C1–O1 = 3.11 Å. The activation energy for this passage was estimated to be ∼20 kcal mol^–1. These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the <i>N</i>-acetamino group of the donor participates in the catalytic mechanism

Substrate-Assisted Catalytic Mechanism of <i>O</i>‑GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding <i>O</i>-linked <i>N</i>-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of <i>O</i>-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer’s disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase <i>O</i>-GlcNAc transferase (uridine diphospho-<i>N</i>-acetylglucosamine:polypeptide β-<i>N</i>-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM­(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme–substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11 326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S_N2-like mechanism, in which a nucleophilic attack by O_Ser, facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1–O_Ser = 1.92 Å and C1–O1 = 3.11 Å. The activation energy for this passage was estimated to be ∼20 kcal mol^–1. These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the <i>N</i>-acetamino group of the donor participates in the catalytic mechanism

In Silico Mutagenesis and Docking Study of <i>Ralstonia solanacearum</i> RSL Lectin: Performance of Docking Software To Predict Saccharide Binding

In this study, in silico mutagenesis and docking in <i>Ralstonia solanacearum</i> lectin (RSL) were carried out, and the ability of several docking software programs to calculate binding affinity was evaluated. In silico mutation of six amino acid residues (Agr17, Glu28, Gly39, Ala40, Trp76, and Trp81) was done, and a total of 114 in silico mutants of RSL were docked with Me-α-l-fucoside. Our results show that polar residues Arg17 and Glu28, as well as nonpolar amino acids Trp76 and Trp81, are crucial for binding. Gly39 may also influence ligand binding because any mutations at this position lead to a change in the binding pocket shape. The Ala40 residue was found to be the most interesting residue for mutagenesis and can affect the selectivity and/or affinity. In general, the docking software used performs better for high affinity binders and fails to place the binding affinities in the correct order

Substrate-Assisted Catalytic Mechanism of <i>O</i>‑GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding <i>O</i>-linked <i>N</i>-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of <i>O</i>-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer’s disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase <i>O</i>-GlcNAc transferase (uridine diphospho-<i>N</i>-acetylglucosamine:polypeptide β-<i>N</i>-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM­(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme–substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11 326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S_N2-like mechanism, in which a nucleophilic attack by O_Ser, facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1–O_Ser = 1.92 Å and C1–O1 = 3.11 Å. The activation energy for this passage was estimated to be ∼20 kcal mol^–1. These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the <i>N</i>-acetamino group of the donor participates in the catalytic mechanism

Substrate-Assisted Catalytic Mechanism of <i>O</i>‑GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding <i>O</i>-linked <i>N</i>-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of <i>O</i>-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer’s disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase <i>O</i>-GlcNAc transferase (uridine diphospho-<i>N</i>-acetylglucosamine:polypeptide β-<i>N</i>-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM­(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme–substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11 326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S_N2-like mechanism, in which a nucleophilic attack by O_Ser, facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1–O_Ser = 1.92 Å and C1–O1 = 3.11 Å. The activation energy for this passage was estimated to be ∼20 kcal mol^–1. These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the <i>N</i>-acetamino group of the donor participates in the catalytic mechanism

Substrate-Assisted Catalytic Mechanism of <i>O</i>‑GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation

In higher eukaryotes, a variety of proteins are post-translationally modified by adding <i>O</i>-linked <i>N</i>-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of <i>O</i>-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer’s disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase <i>O</i>-GlcNAc transferase (uridine diphospho-<i>N</i>-acetylglucosamine:polypeptide β-<i>N</i>-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM­(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme–substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11 326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S_N2-like mechanism, in which a nucleophilic attack by O_Ser, facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1–O_Ser = 1.92 Å and C1–O1 = 3.11 Å. The activation energy for this passage was estimated to be ∼20 kcal mol^–1. These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the <i>N</i>-acetamino group of the donor participates in the catalytic mechanism