Automated Training of ReaxFF Reactive Force Fields for Energetics of Enzymatic Reactions

Computational studies of the reaction mechanisms of various enzymes are nowadays based almost exclusively on hybrid QM/MM models. Unfortunately, the success of this approach strongly depends on the selection of the QM region, and computational cost is a crucial limiting factor. An interesting alternative is offered by empirical reactive molecular force fields, especially the ReaxFF potential developed by van Duin and co-workers. However, even though an initial parametrization of ReaxFF for biomolecules already exists, it does not provide the desired level of accuracy. We have conducted a thorough refitting of the ReaxFF force field to improve the description of reaction energetics. To minimize the human effort required, we propose a fully automated approach to generate an extensive training set comprised of thousands of different geometries and molecular fragments starting from a few model molecules. Electrostatic parameters were optimized with QM electrostatic potentials as the main target quantity, avoiding excessive dependence on the choice of reference atomic charges and improving robustness and transferability. The remaining force field parameters were optimized using the VD-CMA-ES variant of the CMA-ES optimization algorithm. This method is able to optimize hundreds of parameters simultaneously with unprecedented speed and reliability. The resulting force field was validated on a real enzymatic system, ppGalNAcT2 glycosyltransferase. The new force field offers excellent qualitative agreement with the reference QM/MM reaction energy profile, matches the relative energies of intermediate and product minima almost exactly, and reduces the overestimation of transition state energies by 27–48% compared with the previous parametrization

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

Bioinformatics and Molecular Dynamics Simulation Study of L1 Stalk Non-Canonical rRNA Elements: Kink-Turns, Loops, and Tetraloops

The L1 stalk is a prominent mobile element of the large ribosomal subunit. We explore the structure and dynamics of its non-canonical rRNA elements, which include two kink-turns, an internal loop, and a tetraloop. We use bioinformatics to identify the L1 stalk RNA conservation patterns and carry out over 11.5 μs of MD simulations for a set of systems ranging from isolated RNA building blocks up to complexes of L1 stalk rRNA with the L1 protein and tRNA fragment. We show that the L1 stalk tetraloop has an unusual GNNA or UNNG conservation pattern deviating from major GNRA and YNMG RNA tetraloop families. We suggest that this deviation is related to a highly conserved tertiary contact within the L1 stalk. The available X-ray structures contain only UCCG tetraloops which in addition differ in orientation (<i>anti</i> vs <i>syn</i>) of the guanine. Our analysis suggests that the <i>anti</i> orientation might be a mis-refinement, although even the <i>anti</i> interaction would be compatible with the sequence pattern and observed tertiary interaction. Alternatively, the <i>anti</i> conformation may be a real substate whose population could be pH-dependent, since the guanine <i>syn</i> orientation requires protonation of cytosine in the tertiary contact. In absence of structural data, we use molecular modeling to explore the GCCA tetraloop that is dominant in bacteria and suggest that the GCCA tetraloop is structurally similar to the YNMG tetraloop. Kink-turn Kt-77 is unusual due to its 11-nucleotide bulge. The simulations indicate that the long bulge is a stalk-specific eight-nucleotide insertion into consensual kink-turn only subtly modifying its structural dynamics. We discuss a possible evolutionary role of helix H78 and a mechanism of L1 stalk interaction with tRNA. We also assess the simulation methodology. The simulations provide a good description of the studied systems with the latest bsc0χ_OL3 force field showing improved performance. Still, even bsc0χ_OL3 is unable to fully stabilize an essential sugar-edge H-bond between the bulge and non-canonical stem of the kink-turn. Inclusion of Mg²⁺ ions may deteriorate the simulations. On the other hand, monovalent ions can in simulations readily occupy experimental Mg²⁺ binding sites

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