A theoretical study is presented of the conformational properties of methyl 4-thio-alpha-maltosid... more A theoretical study is presented of the conformational properties of methyl 4-thio-alpha-maltoside. Quantum-chemical PCILO (perturbed configuration interaction with localized orbitals) energy minimization with evaluation of the solvent effect has been used to calculate semi-rigid and relaxed (phi, psi) conformational-energy surfaces in 1,4-dioxane, methanol, dimethyl sulfoxide, and water. The inclusion of molecular flexibility in the conformational analysis of this disaccharide derivative was found to have a significant effect upon the allowed conformational space of the molecule. Calculations revealed the existence of 15 stable conformers having different internal geometries. The relative abundance of these conformers is strongly influenced by solvent. The locations of the global minima and "virtual" conformations vary with solvent and the flexibility of methyl 4-thio-alpha-maltoside significantly decreases from 1,4-dioxane to water. It was found that the conformational properties of methyl 4-thio-alpha-maltoside differ from those of the corresponding 4-oxygenated analogue, methyl alpha-maltoside. The calculated solution behaviour is supported by very good agreement between the theoretical values mean value of 3JC-4,H-1' = 2.67 and mean value of 3JC-1',H-4 = 5.24 Hz calculated as ensemble averages over all conformers on the relaxed map and the experimental values of 2.95 and 5.15 Hz in water solution.
In both the biological functions and industrial applications of carbohydrates, their conformation... more In both the biological functions and industrial applications of carbohydrates, their conformational behavior along with the dynamic fluctuations that their structures experience are of great significance. The elucidation of the three-dimensional characteristics of carbohydrates and carbohydrate-containing molecules is in general performed by a combination of high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Heteronuclear couplings and nuclear Overhauser effect (NOE) data are major tools for structural determination. The angular dependence of carbon-proton coupling constants has been shown to depend also on the pathways involved. Concerning molecular modeling, the goal is to generate the ensemble of structures that are consistent with the experimental data. This approach is illustrated in the galactomannan chain. It consists of the conformational study of two model disaccharides (mannobiose and epimelibiose) of galactomannans through carbon-proton coupling constants and NOE measurements. Experimental data are then compared to results of conformational analysis that predicted the stable conformers in solution. From these results helical conformations of oligomers are generated and are discussed in terms of the influence of the position and number of galactose units on the conformation of the mannan backbone.
ABSTRACT The oligo- and polysaccharide structures observed in samples of biological origin repres... more ABSTRACT The oligo- and polysaccharide structures observed in samples of biological origin represent the products of a large number of enzymes: the workhorses of the biosynthetic pathways involved are the glycosyltransferases, although some steps necessary for the final glycoconjugate structures result from glycosidase or transglycosylase activities. Glycosyltransferases are, as the name suggests, enzymes which transfer sugars from an activated donor (normally a nucleotide sugar or lipid-linked phosphosugar) to an acceptor, which can be a polypeptide, a lipid or another saccharide. These enzymes have been studied by a wide variety of approaches—biochemical, genetic, chemical, crystallographic—in order to determine their occurrence, substrate specificity, biological function, mechanism and three-dimensional structure.
Saccharides play due to their wide variability of conformational alternatives a crucial role in m... more Saccharides play due to their wide variability of conformational alternatives a crucial role in many recognition events including pathogen-cell adhesion and recognition, signaling, inflammatory response and others1. They are ordinarily found as glycoconjugates which are often localized on the cell surface or in the extracellular space. They are synthesized by glycosyltransferases in a process called glycosylation – one of the most common and imporatant enzymatic reaction in nature. This project is focused on human N-acetylglucosaminyltransferases, which add N acetylglucosaminyl residues to substrate. These glycosylation events accompany many physiological and pathological cell processes2. The upregulation of these enzymes may lead to an aberrant glycosylation and pathological modifications. Increased amounts of Lewis X and sialyl Lewis X is typical for cancer cells and the interaction between tumor-associated sialyl Lewis X and the endothelial cells of target organs, is one of the first and crucial steps of the metastasis cascade3. Thus these enzymes represent a prime target for the design of glycosylation inhibitors with the potential to specifically alter the structure of cell surface glycoproteins.
Hybrid quantum mechanics and molecular mechanics (QM/MM) methods have become a powerful tool to p... more 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.
The 2‐methylaminotetrahydropyran was used as a model to study conformational properties of the N‐... more The 2‐methylaminotetrahydropyran was used as a model to study conformational properties of the N‐glycosidic linkage in glycosylamines. Relaxed two‐dimensional conformational (Φ,Ψ) maps in 20 solvents were calculated by a method in which the total energy is divided into the energy of the isolated molecule and the solvation energy. Molecular geometry optimization has been carried out for each conformer using the quantum chemical method PCILO. The calculated variations of the geometry are consistent with the results obtained by the statistical analysis of available experimental data retrieved from the Cambridge Structural Database. The calculated abundances of conformers show that the polarity of the solvent has little effect on the anomeric ratio, and the form having the methylamino group equatorial is favored in all considered solvents.
Glycosylation is one of the important post-translational modifications that provide greater prote... more Glycosylation is one of the important post-translational modifications that provide greater proteomic diversity This event is also critical for a wide range of biological processes, such as cell adhesion, defense mechanism, cell proliferation, cancer metastasis etc through variety of glycoconjugates. These glycoconjugates are formed by glycosyltransferases which add saccharides onto proteins, lipids sugars etc. Here we are exploring the reaction mechanisms of O-GlcNAc transferase (OGTs), where GlcNAc is transferred to –OH group of Ser/Thr of the proteins. Three groups have proposed mechanisms on different crystal structures of OGTs, suggesting different catalytic base to abstract proton from Ser, 1) His498[1], 2) alpha-phosphate[2], and 3) water molecule shunting[3] , but still the process is not clear. In presented study we were trying to confirm one of the proposed mechanisms employing hybrid QM/MM CPMD molecular dynamics. We have modelled all three proposed mechanisms. The enzyme was partitioned for catalytic site as quantum zone and rest of the protein was treated with MM in explicit solvent box. Whole model has approximately 92000 atoms. Observed results suggest that in all the three mechanisms nucleophilic attack is followed by bond dissociation, proton transfer and bond formation. Obtained energy profiles favour reaction mechanism where alpha-phosphate serves as catalytic base.
The stereochemistry of methoxy(methylthio)methane - a model of the thioacetal segment in thiosacc... more The stereochemistry of methoxy(methylthio)methane - a model of the thioacetal segment in thiosaccharides - has been studied by semiempirical quantum -chemical methods. The conformational map of the rotation around the central C-O and C-S bonds has been calculated by the PCILO method. Decomposition of the energy for the rotation around the C-O (C-S)bond (the methyl group being fixed in the antiperiplanar position) in terms of the Fourier series gives the values 7.12 (2.53) kJ mol-1 for the respective rotational barrier. The geometry of five stable conformers, whose stability decreases in the order (sc,sc) > (ap,sc) > (-ac,sc) > (ap,ap), has been optimized by the MNDO method. Ratio of the individual conformers in an isolated molecule calculated on the basis of the MNDO energies is 92 : 5 : 2 : 1 : 0, the mean dipole moment being 3.6 . 10-30 Cm. The solvent effect has been calculated by a method involving electrostatic and dispersion interactions and cavity contribution. Population of the conformers in diluted solutions depends on nature of the solvent, proportion of the antiperiplanar positions being increased with increasing polarity. The calculated ratios of the conformers are 85 : 8 : 3 : 4 : 0 and 63 : 14 : 7 : 16 : 0 in acetonitrile and water, respectively. The calculated values of the Gibbs energies have also been used for assessment of magnitude of anomeric and exo-anomeric effects in α- and β-1-thiopyranosides and α- and β-5-thiopyranosides.
A theoretical study is presented of the conformational properties of methyl 4-thio-alpha-maltosid... more A theoretical study is presented of the conformational properties of methyl 4-thio-alpha-maltoside. Quantum-chemical PCILO (perturbed configuration interaction with localized orbitals) energy minimization with evaluation of the solvent effect has been used to calculate semi-rigid and relaxed (phi, psi) conformational-energy surfaces in 1,4-dioxane, methanol, dimethyl sulfoxide, and water. The inclusion of molecular flexibility in the conformational analysis of this disaccharide derivative was found to have a significant effect upon the allowed conformational space of the molecule. Calculations revealed the existence of 15 stable conformers having different internal geometries. The relative abundance of these conformers is strongly influenced by solvent. The locations of the global minima and "virtual" conformations vary with solvent and the flexibility of methyl 4-thio-alpha-maltoside significantly decreases from 1,4-dioxane to water. It was found that the conformational properties of methyl 4-thio-alpha-maltoside differ from those of the corresponding 4-oxygenated analogue, methyl alpha-maltoside. The calculated solution behaviour is supported by very good agreement between the theoretical values mean value of 3JC-4,H-1' = 2.67 and mean value of 3JC-1',H-4 = 5.24 Hz calculated as ensemble averages over all conformers on the relaxed map and the experimental values of 2.95 and 5.15 Hz in water solution.
In both the biological functions and industrial applications of carbohydrates, their conformation... more In both the biological functions and industrial applications of carbohydrates, their conformational behavior along with the dynamic fluctuations that their structures experience are of great significance. The elucidation of the three-dimensional characteristics of carbohydrates and carbohydrate-containing molecules is in general performed by a combination of high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Heteronuclear couplings and nuclear Overhauser effect (NOE) data are major tools for structural determination. The angular dependence of carbon-proton coupling constants has been shown to depend also on the pathways involved. Concerning molecular modeling, the goal is to generate the ensemble of structures that are consistent with the experimental data. This approach is illustrated in the galactomannan chain. It consists of the conformational study of two model disaccharides (mannobiose and epimelibiose) of galactomannans through carbon-proton coupling constants and NOE measurements. Experimental data are then compared to results of conformational analysis that predicted the stable conformers in solution. From these results helical conformations of oligomers are generated and are discussed in terms of the influence of the position and number of galactose units on the conformation of the mannan backbone.
ABSTRACT The oligo- and polysaccharide structures observed in samples of biological origin repres... more ABSTRACT The oligo- and polysaccharide structures observed in samples of biological origin represent the products of a large number of enzymes: the workhorses of the biosynthetic pathways involved are the glycosyltransferases, although some steps necessary for the final glycoconjugate structures result from glycosidase or transglycosylase activities. Glycosyltransferases are, as the name suggests, enzymes which transfer sugars from an activated donor (normally a nucleotide sugar or lipid-linked phosphosugar) to an acceptor, which can be a polypeptide, a lipid or another saccharide. These enzymes have been studied by a wide variety of approaches—biochemical, genetic, chemical, crystallographic—in order to determine their occurrence, substrate specificity, biological function, mechanism and three-dimensional structure.
Saccharides play due to their wide variability of conformational alternatives a crucial role in m... more Saccharides play due to their wide variability of conformational alternatives a crucial role in many recognition events including pathogen-cell adhesion and recognition, signaling, inflammatory response and others1. They are ordinarily found as glycoconjugates which are often localized on the cell surface or in the extracellular space. They are synthesized by glycosyltransferases in a process called glycosylation – one of the most common and imporatant enzymatic reaction in nature. This project is focused on human N-acetylglucosaminyltransferases, which add N acetylglucosaminyl residues to substrate. These glycosylation events accompany many physiological and pathological cell processes2. The upregulation of these enzymes may lead to an aberrant glycosylation and pathological modifications. Increased amounts of Lewis X and sialyl Lewis X is typical for cancer cells and the interaction between tumor-associated sialyl Lewis X and the endothelial cells of target organs, is one of the first and crucial steps of the metastasis cascade3. Thus these enzymes represent a prime target for the design of glycosylation inhibitors with the potential to specifically alter the structure of cell surface glycoproteins.
Hybrid quantum mechanics and molecular mechanics (QM/MM) methods have become a powerful tool to p... more 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.
The 2‐methylaminotetrahydropyran was used as a model to study conformational properties of the N‐... more The 2‐methylaminotetrahydropyran was used as a model to study conformational properties of the N‐glycosidic linkage in glycosylamines. Relaxed two‐dimensional conformational (Φ,Ψ) maps in 20 solvents were calculated by a method in which the total energy is divided into the energy of the isolated molecule and the solvation energy. Molecular geometry optimization has been carried out for each conformer using the quantum chemical method PCILO. The calculated variations of the geometry are consistent with the results obtained by the statistical analysis of available experimental data retrieved from the Cambridge Structural Database. The calculated abundances of conformers show that the polarity of the solvent has little effect on the anomeric ratio, and the form having the methylamino group equatorial is favored in all considered solvents.
Glycosylation is one of the important post-translational modifications that provide greater prote... more Glycosylation is one of the important post-translational modifications that provide greater proteomic diversity This event is also critical for a wide range of biological processes, such as cell adhesion, defense mechanism, cell proliferation, cancer metastasis etc through variety of glycoconjugates. These glycoconjugates are formed by glycosyltransferases which add saccharides onto proteins, lipids sugars etc. Here we are exploring the reaction mechanisms of O-GlcNAc transferase (OGTs), where GlcNAc is transferred to –OH group of Ser/Thr of the proteins. Three groups have proposed mechanisms on different crystal structures of OGTs, suggesting different catalytic base to abstract proton from Ser, 1) His498[1], 2) alpha-phosphate[2], and 3) water molecule shunting[3] , but still the process is not clear. In presented study we were trying to confirm one of the proposed mechanisms employing hybrid QM/MM CPMD molecular dynamics. We have modelled all three proposed mechanisms. The enzyme was partitioned for catalytic site as quantum zone and rest of the protein was treated with MM in explicit solvent box. Whole model has approximately 92000 atoms. Observed results suggest that in all the three mechanisms nucleophilic attack is followed by bond dissociation, proton transfer and bond formation. Obtained energy profiles favour reaction mechanism where alpha-phosphate serves as catalytic base.
The stereochemistry of methoxy(methylthio)methane - a model of the thioacetal segment in thiosacc... more The stereochemistry of methoxy(methylthio)methane - a model of the thioacetal segment in thiosaccharides - has been studied by semiempirical quantum -chemical methods. The conformational map of the rotation around the central C-O and C-S bonds has been calculated by the PCILO method. Decomposition of the energy for the rotation around the C-O (C-S)bond (the methyl group being fixed in the antiperiplanar position) in terms of the Fourier series gives the values 7.12 (2.53) kJ mol-1 for the respective rotational barrier. The geometry of five stable conformers, whose stability decreases in the order (sc,sc) > (ap,sc) > (-ac,sc) > (ap,ap), has been optimized by the MNDO method. Ratio of the individual conformers in an isolated molecule calculated on the basis of the MNDO energies is 92 : 5 : 2 : 1 : 0, the mean dipole moment being 3.6 . 10-30 Cm. The solvent effect has been calculated by a method involving electrostatic and dispersion interactions and cavity contribution. Population of the conformers in diluted solutions depends on nature of the solvent, proportion of the antiperiplanar positions being increased with increasing polarity. The calculated ratios of the conformers are 85 : 8 : 3 : 4 : 0 and 63 : 14 : 7 : 16 : 0 in acetonitrile and water, respectively. The calculated values of the Gibbs energies have also been used for assessment of magnitude of anomeric and exo-anomeric effects in α- and β-1-thiopyranosides and α- and β-5-thiopyranosides.
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Papers by Igor Tvaroška