Stepwise Catalytic Mechanism via Short-Lived Intermediate Inferred from Combined QM/MM MERP and PES Calculations on Retaining Glycosyltransferase ppGalNAcT2

The glycosylation of cell surface proteins plays a crucial role in a multitude of biological processes, such as cell adhesion and recognition. To understand the process of protein glycosylation, the reaction mechanisms of the participating enzymes need to be known. However, the reaction mechanism of retaining glycosyltransferases has not yet been sufficiently explained. Here we investigated the catalytic mechanism of human isoform 2 of the retaining glycosyltransferase polypeptide UDP-GalNAc transferase by coupling two different QM/MM-based approaches, namely a potential energy surface scan in two distance difference dimensions and a minimum energy reaction path optimisation using the Nudged Elastic Band method. Potential energy scan studies often suffer from inadequate sampling of reactive processes due to a predefined scan coordinate system. At the same time, path optimisation methods enable the sampling of a virtually unlimited number of dimensions, but their results cannot be unambiguously interpreted without knowledge of the potential energy surface. By combining these methods, we have been able to eliminate the most significant sources of potential errors inherent to each of these approaches. The structural model is based on the crystal structure of human isoform 2. In the QM/MM method, the QM region consists of 275 atoms, the remaining 5776 atoms were in the MM region. We found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The optimized transition state for the reaction is 13.8 kcal/mol higher in energy than the reactant while the energy of the product complex is 6.7 kcal/mol lower. During the process of nucleophilic attack, a proton is synchronously transferred to the leaving phosphate. The presence of a short-lived metastable oxocarbenium intermediate is likely, as indicated by the reaction energy profiles obtained using high-level density functionals

Molecular Modeling Insights into the Structure and Behavior of Integrins: A Review

Integrins are heterodimeric glycoproteins crucial to the physiology and pathology of many biological functions. As adhesion molecules, they mediate immune cell trafficking, migration, and immunological synapse formation during inflammation and cancer. The recognition of the vital roles of integrins in various diseases revealed their therapeutic potential. Despite the great effort in the last thirty years, up to now, only seven integrin-based drugs have entered the market. Recent progress in deciphering integrin functions, signaling, and interactions with ligands, along with advancement in rational drug design strategies, provide an opportunity to exploit their therapeutic potential and discover novel agents. This review will discuss the molecular modeling methods used in determining integrins’ dynamic properties and in providing information toward understanding their properties and function at the atomic level. Then, we will survey the relevant contributions and the current understanding of integrin structure, activation, the binding of essential ligands, and the role of molecular modeling methods in the rational design of antagonists. We will emphasize the role played by molecular modeling methods in progress in these areas and the designing of integrin antagonists

Plant Xyloglucan Xyloglucosyl Transferases and the Cell Wall Structure: Subtle but Significant

Plant xyloglucan xyloglucosyl transferases or xyloglucan endo-transglycosylases (XET; EC 2.4.1.207) catalogued in the glycoside hydrolase family 16 constitute cell wall-modifying enzymes that play a fundamental role in the cell wall expansion and re-modelling. Over the past thirty years, it has been established that XET enzymes catalyse homo-transglycosylation reactions with xyloglucan (XG)-derived substrates and hetero-transglycosylation reactions with neutral and charged donor and acceptor substrates other than XG-derived. This broad specificity in XET isoforms is credited to a high degree of structural and catalytic plasticity that has evolved ubiquitously in algal, moss, fern, basic Angiosperm, monocot, and eudicot enzymes. These XET isoforms constitute gene families that are differentially expressed in tissues in time- and space-dependent manners during plant growth and development, and in response to biotic and abiotic stresses. Here, we discuss the current state of knowledge of broad specific plant XET enzymes and how their inherently carbohydrate-based transglycosylation reactions tightly link with structural diversity that underlies the complexity of plant cell walls and their mechanics. Based on this knowledge, we conclude that multi- or poly-specific XET enzymes are widespread in plants to allow for modifications of the cell wall structure in muro, a feature that implements the multifaceted roles in plant cells

Definition of the acceptor substrate binding specificity in plant xyloglucan endotransglycosylases using computational chemistry

The visualisation of the MD trajectory of the XG-OS7 donor and Xyl-OS4 acceptor substrates in the active site of PttXET16A reveals the instability of the acceptor. After 20 ns the Xyl-OS4 chain changes its position and approaches the loop with S257. However, unlike Cello-OS4 this interaction does not stabilise the acceptor. After 50 ns the first signs of destabilisation of Xyl-OS4 can be observed, and after 460 ns its chain moves into a position that is unfavourable for glycosidic bond formation. Conversely, the Xyl-OS4 acceptor in the active site of TmXET6.3 remains stable during the duration of the MD simulation.</p

Reaction catalysed by the ppGalNAcT2 glycosyltransferase.

<p>The names of atoms used to define PES scan coordinates are set in bold.</p

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

Parts of conserved enzyme residues included in QM region.

<p>Parts of conserved enzyme residues included in QM region.</p

Basic parameters of stationary point structures.

<p>Basic parameters of stationary point structures.</p

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