Glycosylation Modulates the Structure and Functions of Collagen: A Review.

Collagens are fundamental constituents of the extracellular matrix and are the most abundant proteins in mammals. Collagens belong to the family of fibrous or fiber-forming proteins that self-assemble into fibrils that define their mechanical properties and biological functions. Up to now, 28 members of the collagen superfamily have been recognized. Collagen biosynthesis occurs in the endoplasmic reticulum, where specific post-translational modification-glycosylation-is also carried out. The glycosylation of collagens is very specific and adds ß-d-galactopyranose and ß-d-Glcp-(1→2)-d-Galp disaccharide through ß-O-linkage to hydroxylysine. Several glycosyltransferases, namely COLGALT1, COLGALT2, LH3, and PGGHG glucosidase, were associated the with glycosylation of collagens, and recently, the crystal structure of LH3 has been solved. Although not fully understood, it is clear that the glycosylation of collagens influences collagen secretion and the alignment of collagen fibrils. A growing body of evidence also associates the glycosylation of collagen with its functions and various human diseases. Recent progress in understanding collagen glycosylation allows for the exploitation of its therapeutic potential and the discovery of new agents. This review will discuss the relevant contributions to understanding the glycosylation of collagens. Then, glycosyltransferases involved in collagen glycosylation, their structure, and catalytic mechanism will be surveyed. Furthermore, the involvement of glycosylation in collagen functions and collagen glycosylation-related diseases will be discussed.

How E-, L-, and P-Selectins Bind to sLex and PSGL-1: A Quantification of Critical Residue Interactions.

Selectins and their ability to interact with specific ligands are a cornerstone in cell communication. Over the last three decades, a considerable wealth of experimental and molecular modeling insights into their structure and modus operandi were gathered. Nonetheless, explaining the role of individual selectin residues on a quantitative level remained elusive, despite its importance in understanding the structure-function relationship in these molecules and designing their inhibitors. This work explores essential interactions of selectin-ligand binding, employing a multiscale approach that combines molecular dynamics, quantum-chemical calculations, and residue interaction network models. Such an approach successfully reproduces most of the experimental findings. It proves to be helpful, with the potential for becoming an established tool for quantitative predictions of residue contribution to the binding of biomolecular complexes. The results empower us to quantify the importance of particular residues and functional groups in the protein-ligand interface and to pinpoint differences in molecular recognition by the three selectins. We show that mutations in the E-, L-, and P-selectins, e.g., different residues in positions 46, 85, 97, and 107, present a crucial difference in how the ligand is engaged. We assess the role of sulfation of tyrosine residues in PSGL-1 and suggest that TyrSO3- in position 51 interacting with Arg85 in P-selectin is a significant factor in the increased affinity of P-selectin to PSGL-1 compared to E- and L-selectins. We propose an original pharmacophore targeting five essential PSGL-binding sites based on the analysis of the selectin···PSGL-1 interactions.

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.

The catalytic reaction mechanism of tyrosylprotein sulfotransferase-1.

Tyrosine sulfation alters the biological activity of many proteins involved in different physiological and pathophysiological conditions, such as non-specific immune reaction, response to inflammation and ischemia, targeting of leukocytes and stem cells, or the formation of cancer metastases. Tyrosine sulfation is catalyzed by the enzymes tyrosylprotein sulfotransferases (TPST). In this study, we used QM/MM Car-Parrinello metadynamics simulations together with QM/MM potential energy calculations to investigate the catalytic mechanism of isoform TPST-1. The structural changes along the reaction coordinate are analyzed and discussed. Furthermore, both the methods supported the SN2 type of catalytic mechanism. The reaction barrier obtained from CPMD metadynamics was 12.8 kcal mol-1, and the potential energy scan led to reaction barriers of 11.6 kcal mol-1 and 13.7 kcal mol-1 with the B3LYP and OPBE functional, respectively. The comparison of the two methods (metadynamics and potential energy scan) may be helpful for future mechanistic studies. The insight into the reaction mechanism of TPST-1 might help with the rational design of transition-state TPST inhibitors.

Pan-selectin inhibitors as potential therapeutics for COVID-19 treatment: in silico screening study.

Coronavirus disease 2019 (COVID-19) has spread rapidly throughout the globe. The spectrum of disease is broad but among hospitalized patients with COVID-19, respiratory failure from acute respiratory distress syndrome is the leading cause of mortality. There is an urgent need for an effective treatment. The current focus has been developing novel therapeutics, including antivirals, protease inhibitors, vaccines and targeting the overactive cytokine response with anti-cytokine therapy. The overproduction of early response proinflammatory cytokines results in what has been described as a "cytokine storm" is leading eventually to death when the cells fail to terminate the inflammatory response. Accumulating evidence shows that inflammatory cytokines induce selectin ligands that play a crucial role in the pathogenesis of inflammatory diseases by mediating leukocyte migration from the blood into the tissue. Thus, the selectins and selectin ligands represent a promising therapeutic target for the treatment of COVID-19. In this paper, potential pan-selectin inhibitors were identified employing a virtual screening using a docking procedure. For this purpose, the Asinex and ZINC databases of ligands, including approved drugs, biogenic compounds and glycomimetics, altogether 923,602 compounds, were screened against the P-, L- and E-selectin. At first, the experimentally confirmed inhibitors were docked into all three selectins' carbohydrate recognition domains to assess the suitability of the screening procedure. Finally, based on the evaluation of ligands binding, we propose 10 purchasable pan-selectin inhibitors to develop COVID-19 therapeutics.

Catalytic Mechanism of Processive GlfT2: Transition Path Sampling Investigation of Substrate Translocation.

We applied the transition path sampling (TPS) method to study the translocation step of the catalytic mechanism of galactofuranosyl transferase 2 (GlfT2). Using TPS in the field of enzymatic reactions is still relatively rare, and we show its effectiveness on this enzymatic system. We decipher an unknown mechanism of the translocation step and, thus, provide a complete understanding of the catalytic mechanism of GlfT2 at the atomistic level. The GlfT2 enzyme is involved in the formation of the mycobacterial cell wall and transfers galactofuranose (Galf) from UDP-Galf onto a growing acceptor Galf chain. The biosynthesis of the galactan chain is accomplished in a processive manner, with the growing acceptor substrate remaining bound to GlfT2. The glycosidic bond formed by GlfT2 between the two Galf residues alternates between ß-(1-6) and ß-(1-5) linkages. The translocation of the growing galactan between individual additions of Galf residues is crucial for the function of GlfT2. Analysis of unbiased trajectory ensembles revealed that the translocation proceeds differently depending on the glycosidic linkage between the last two Galf residues. We also showed that the protonation state of the catalytic residue Asp372 significantly influences the translocation. Approximate transition state structures and potential energy reaction barriers of the translocation process were determined. The calculated potential reaction barriers in the range of 6-14 kcal/mol show that the translocation process is not the rate-limiting step in galactan biosynthesis.

Selectins-The Two Dr. Jekyll and Mr. Hyde Faces of Adhesion Molecules-A Review.

Selectins belong to a group of adhesion molecules that fulfill an essential role in immune and inflammatory responses and tissue healing. Selectins are glycoproteins that decode the information carried by glycan structures, and non-covalent interactions of selectins with these glycan structures mediate biological processes. The sialylated and fucosylated tetrasaccharide sLex is an essential glycan recognized by selectins. Several glycosyltransferases are responsible for the biosynthesis of the sLex tetrasaccharide. Selectins are involved in a sequence of interactions of circulated leukocytes with endothelial cells in the blood called the adhesion cascade. Recently, it has become evident that cancer cells utilize a similar adhesion cascade to promote metastases. However, like Dr. Jekyll and Mr. Hyde's two faces, selectins also contribute to tissue destruction during some infections and inflammatory diseases. The most prominent function of selectins is associated with the initial stage of the leukocyte adhesion cascade, in which selectin binding enables tethering and rolling. The first adhesive event occurs through specific non-covalent interactions between selectins and their ligands, with glycans functioning as an interface between leukocytes or cancer cells and the endothelium. Targeting these interactions remains a principal strategy aimed at developing new therapies for the treatment of immune and inflammatory disorders and cancer. In this review, we will survey the significant contributions to and the current status of the understanding of the structure of selectins and the role of selectins in various biological processes. The potential of selectins and their ligands as therapeutic targets in chronic and acute inflammatory diseases and cancer will also be discussed. We will emphasize the structural characteristic of selectins and the catalytic mechanisms of glycosyltransferases involved in the biosynthesis of glycan recognition determinants. Furthermore, recent achievements in the synthesis of selectin inhibitors will be reviewed with a focus on the various strategies used for the development of glycosyltransferase inhibitors, including substrate analog inhibitors and transition state analog inhibitors, which are based on knowledge of the catalytic mechanism.

Atomistic simulation of carbohydrate-protein complex formation: Hevein-32 domain.

Interactions between proteins and their small molecule ligands are of great importance for the process of drug design. Here we report an unbiased molecular dynamics simulation of systems containing hevein domain (HEV32) with N-acetylglucosamine mono-, di- or trisaccharide. Carbohydrate molecules were placed outside the binding site. Three of six simulations (6 × 2 µs) led to binding of a carbohydrate ligand into the binding mode in agreement with the experimentally determined structure. Unbinding was observed in one simulation (monosaccharide). There were no remarkable intermediates of binding for mono and disaccharide. Trisaccharide binding was initiated by formation of carbohydrate-aromatic CH/π interactions. Our results indicate that binding of ligands followed the model of conformational selection because the conformation of the protein ready for ligand binding was observed before the binding. This study extends the concept of docking by dynamics on carbohydrate-protein interactions.

Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site.

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.

How Mycobacterium tuberculosis Galactofuranosyl Transferase†2 (GlfT2) Generates Alternating ß-(1-6) and ß-(1-5) Linkages: A QM/MM Molecular Dynamics Study of the Chemical Steps.

Mycobacterium tuberculosis features a unique cell wall that protects the bacterium from the external environment. Disruption of the cell wall assembly is a promising direction for novel anti-tuberculotic drugs. A key component of the cell wall is galactan, a polysaccharide chain composed of galactofuranose (Galf) units connected by alternating ß-(1-5) and ß-(1-6) linkages. The majority of the galactan chain is biosynthesized by a bifunctional enzyme-galactofuranosyl transferase 2 (GlfT2). GlfT2 catalyzes two reactions: the formation of ß-(1-5) and ß-(1-6) linkages. It was suggested that the enzyme acts through a processive mechanism until it adds 30-35 Galf units in a single active site. We applied a QM/MM string method coupled with ab initio molecular dynamics simulations to study the two reactions catalyzed by GlfT2. We showed that both reactions proceed very similarly and feature similar transition-state structures. We also present novel information about the ring puckering behavior of the five-membered furanose ring during the glycosyltransferase reaction and a calculated transition-state structure with galactose in a furanose form that may be used as a guide for the rational design of very specific and extremely potent inhibitors, that is, transition-state analogues, for GlfT2. Due to the absence of a furanose form of galactose in humans, transition-state-analogous inhibitors represent an attractive scaffold for the development of novel antibacterial drugs.

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.

First-Principles Interaction Analysis Assessment of the Manganese Cation in the Catalytic Activity of Glycosyltransferases.

The energetic effect of water substitution reactions in hexacoordinated [Mn(H2O)6-nLzn]2+nz complexes with L = methanol, formic acid, formamide, formate, imidazole, and diphosphate is quantitatively analyzed at the MP2/triple-ζ level of theory. Subsequently, the state-of-the-art open shell symmetry-adapted perturbation theory (SAPT) analysis of the interaction energies of Mn2+···ligand dimers with selected O-, S-, and N-binding ligands is presented and compared to similar interactions of Mg2+ and Zn2+ ions. We find that the induction energies in the dimers with manganese are almost twice as large as in dimers with magnesium. The total interaction energies rise in the order Mn2+ < Mg2+ < Zn2+. The calculations of the Mn2+ → Mg2+ replacement reaction suggest that metal-dependent glycosyltransferases influence the binding preference of Mn2+ over Mg2+ by inserting amino acids that coordinate the metal via nitrogen or sulfur into their active site.

Different QM/MM Approaches To Elucidate Enzymatic Reactions: Case Study on ppGalNAcT2.

Hybrid QM/MM computational studies can provide invaluable insight into the mechanisms of enzymatic reactions that can be exploited for rational drug design. Various approaches are available for such studies. However, their strengths and weaknesses may not be immediately apparent. Using the retaining glycosyltransferase ppGalNAcT2 as a case study, we compare different methodologies used to obtain reaction paths and transition state information. Ab Initio MD using CPMD coupled with the String Method is used to derive the minimum free energy reaction path. The geometrical features of the free energy path, especially around the transition state, agree with the minimum potential energy path obtained by the much less computationally expensive Nudged Elastic Band method. The barrier energy, however, differs by 8 kcal/mol. The free energy surface generated by metadynamics provides a rough overview of the reaction and can confirm the physical relevance of optimized paths or provide an initial guess for path optimization methods. Calculations of enzymatic reactions are usually performed at best at the DFT level of theory. A comparison of widely used functionals with high-level DLPNO-CCSD(T)/CBS data on the potential energy profile serves as a validation of the usability of DFT for this type of enzymatic reaction. The M06-2X meta-hybrid functional in particular matches the DLPNO-CCSD(T)/CBS reference extremely well with errors within 1 kcal/mol. However, even pure-GGA functional OPBE provides sufficient accuracy for this type of study.

Using DFT methodology for more reliable predictive models: Design of inhibitors of Golgi α-Mannosidase II.

Human Golgi α-mannosidase II (GMII), a zinc ion co-factor dependent glycoside hydrolase (E.C.3.2.1.114), is a pharmaceutical target for the design of inhibitors with anti-cancer activity. The discovery of an effective inhibitor is complicated by the fact that all known potent inhibitors of GMII are involved in unwanted co-inhibition with lysosomal α-mannosidase (LMan, E.C.3.2.1.24), a relative to GMII. Routine empirical QSAR models for both GMII and LMan did not work with a required accuracy. Therefore, we have developed a fast computational protocol to build predictive models combining interaction energy descriptors from an empirical docking scoring function (Glide-Schrödinger), Linear Interaction Energy (LIE) method, and quantum mechanical density functional theory (QM-DFT) calculations. The QSAR models were built and validated with a library of structurally diverse GMII and LMan inhibitors and non-active compounds. A critical role of QM-DFT descriptors for the more accurate prediction abilities of the models is demonstrated. The predictive ability of the models was significantly improved when going from the empirical docking scoring function to mixed empirical-QM-DFT QSAR models (Q(2)=0.78-0.86 when cross-validation procedures were carried out; and R(2)=0.81-0.83 for a testing set). The average error for the predicted ΔGbind decreased to 0.8-1.1kcalmol(-1). Also, 76-80% of non-active compounds were successfully filtered out from GMII and LMan inhibitors. The QSAR models with the fragmented QM-DFT descriptors may find a useful application in structure-based drug design where pure empirical and force field methods reached their limits and where quantum mechanics effects are critical for ligand-receptor interactions. The optimized models will apply in lead optimization processes for GMII drug developments.

Three-dimensional homology model of GlcNAc-TV glycosyltransferase.

The enzyme UDP-N-acetylglucosamine: α-d-mannoside ß-1-6 N-acetylglucosaminyltransferase V (GnT-V) catalyzes the transfer of GlcNAc from the UDP-GlcNAc donor to the α-1-6-linked mannose of the trimannosyl core structure of glycoproteins to produce the ß-1-6-linked branching of N-linked oligosaccharides. ß-1-6-GlcNAc-branched N-glycans are associated with cancer growth and metastasis. Therefore, the inhibition of GnT-V represents a key target for anti-cancer drug development. However, the development of potent and specific inhibitors of GnT-V is hampered by the lack of information on the three-dimensional structure of the enzyme and on the binding characteristics of its substrates. Here we present the first 3D structure of GnT-V as a result of homology modeling. Various alignment methods, docking the donor and acceptor substrates, and molecular dynamics simulation were used to construct seven homology models of GnT-V and characterize the binding of its substrates. The best homology model is consistent with available experimental data. The three-dimensional model, the structure of the enzyme catalytic site and binding information obtained for the donor and acceptor can be useful in studies of the catalytic mechanism and design of inhibitors of GnT-V.

Ab initio modelling of the anomeric and exo anomeric effects in 2-methoxytetrahydropyran and 2-methoxythiane corrected for intramolecular BSSE.

Accurate ab initio calculations including basis set limit (BSL) extrapolations, removal of intramolecular basis set superposition error (BSSE), solvent effect corrections, and thermal effects have been carried out to compare the structure and the anomeric and exo-anomeric effect in 2-methoxytetrahydropyran and 2-methoxythiane. The effect of intramolecular BSSE on the energetics was outlined for the first time in these types of compounds. It was found that both title compounds show comparable behaviour with respect to BSSE. The energy gap between the axial and equatorial form of 2-methoxythiane is reduced by 0.23 kcal mol(-1) due to the BSSE correction at the MP2/aug-cc-pVTZ level of theory, and in 2-methoxytetrahydropyran it is reduced by 0.21 kcal mol(-1). The intramolecular BSSE influenced also the energy differences between the gauche and trans conformers in both compounds. Energy decomposition analysis (EDA) reveals that the dominant destabilising interaction is repulsion and its primary stabilizing counterpart is the polarization interaction.

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 simulations of hevein/(GlcNAc)3 complex with weakened OH/O and CH/π hydrogen bonds: implications for their role in complex stabilization.

Carbohydrate-protein complexes are often characterized by interactions via aromatic amino acid residues. Several mechanisms have been proposed to explain these stacking-like interactions between pyranose sugars and aromatic moieties. The physical basis of these interactions is being explained as either dispersion CH/π or hydrophobic. In order to elucidate the nature of these interactions, we performed a series of molecular dynamics simulation of hevein domain (HEV32) in complex with (ß-D-GlcNAc)3. Selected OH/O and CH/π hydrogen bonds involved in carbohydrate recognition were artificially weakened in 100 ns molecular dynamics simulations. Separate weakening of either OH/O or CH/π hydrogen bonds was not sufficient to destabilize the complex. This indicates that other effects, not solely CH/π dispersion interactions, contribute significantly to the stability of the complex. Significant destabilization of complexes was reached only by simultaneous weakening of OH/O and CH/π hydrogen bonds. This also shows that classical hydrogen bonds and CH/π interactions are working in concert to stabilize this carbohydrate-protein test case.

Exploring reaction pathways for O-GlcNAc transferase catalysis. A string method study.

The inverting O-GlcNAc glycosyltransferase (OGT) is an important post-translation enzyme, which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine (UDP-GlcNAc) to the hydroxyl group of the Ser/Thr of cytoplasmic, nuclear, and mitochondrial proteins. In the past, three different catalytic bases were proposed for the reaction: His498, α-phosphate, and Asp554. In this study, we used hybrid quantum mechanics/molecular mechanics (QM/MM) Car-Parrinello molecular dynamics to investigate reaction paths using α-phosphate and Asp554 as the catalytic bases. The string method was used to calculate the free-energy reaction profiles of the tested mechanisms. During the investigations, an additional mechanism was observed. In this mechanism, a proton is transferred to α-phosphate via a water molecule. Our calculations show that the mechanism with α-phosphate acting as the base is favorable. This reaction has a rate-limiting free-energy barrier of 23.5 kcal/mol, whereas reactions utilizing Asp554 and water-assisted α-phosphate have barriers of 41.7 and 40.9 kcal/mol, respectively. Our simulations provide a new insight into the catalysis of OGT and may thus guide rational drug design of transition-state analogue inhibitors with potential therapeutic use.

QM/MM methods for studying enzymatic reactions of glycosyltransferases.

Hybrid quantum mechanics and molecular mechanics (QM/MM) methods have become a powerful tool to provide an accurate and effective description of complex biological systems. The QM treatment of the electronic structure of an active site region and the rest of the enzyme by molecular mechanics allows enzymatic reaction to being modeled with including the impact of environment. Different reaction pathways of the enzymatic mechanism can be tested--transition states (TS) and intermediates characterized using QM/MM methods, leading to significant advances in understanding enzymatic reactions. This chapter discusses the ideas and the setting up of the structural and computational models for calculations with QM/MM software. The use of QM/MM methodology is also illustrated using the case of the inverting glycosyltransferase GnT-I.