RESUMEN
Structural determinants of substrate recognition remain inadequately defined in broad specific cell-wall modifying enzymes, termed xyloglucan xyloglucosyl transferases (XETs). Here, we investigate the Tropaeolum majus seed TmXET6.3 isoform, a member of the GH16_20 subfamily of the GH16 network. This enzyme recognises xyloglucan (XG)-derived donors and acceptors, and a wide spectrum of other chiefly saccharide substrates, although it lacks the activity with homogalacturonan (pectin) fragments. We focus on defining the functionality of carboxyl-terminal residues in TmXET6.3, which extend acceptor binding regions in the GH16_20 subfamily but are absent in the related GH16_21 subfamily. Site-directed mutagenesis using double to quintuple mutants in the carboxyl-terminal region - substitutions emulated on barley XETs recognising the XG/penta-galacturonide acceptor substrate pair - demonstrated that this activity could be gained in TmXET6.3. We demonstrate the roles of semi-conserved Arg238 and Lys237 residues, introducing a net positive charge in the carboxyl-terminal region (which complements a negative charge of the acidic penta-galacturonide) for the transfer of xyloglucan fragments. Experimental data, supported by molecular modelling of TmXET6.3 with the XG oligosaccharide donor and penta-galacturonide acceptor substrates, indicated that they could be accommodated in the active site. Our findings support the conclusion on the significance of positively charged residues at the carboxyl terminus of TmXET6.3 and suggest that a broad specificity could be engineered via modifications of an acceptor binding site. The definition of substrate specificity in XETs should prove invaluable for defining the structure, dynamics, and function of plant cell walls, and their metabolism; these data could be applicable in various biotechnologies.
Asunto(s)
Aminoácidos , Glicosiltransferasas , Especificidad por Sustrato , Glicosiltransferasas/metabolismo , Aminoácidos/metabolismo , Células Vegetales/metabolismo , Pared Celular/metabolismo , Xilanos/metabolismoRESUMEN
Mycobacterium tuberculosis, one of the deadliest pathogens in human history, is distinguished by a unique, multilayered cell wall, which offers the bacterium a high level of protection from the attacks of the host immune system. The primary structure of the cell wall core, composed of covalently linked peptidoglycan, branched heteropolysaccharide arabinogalactan, and mycolic acids, is well known, and numerous enzymes involved in the biosynthesis of its components are characterized. The cell wall biogenesis takes place at both cytoplasmic and periplasmic faces of the plasma membrane, and only recently some of the specific transport systems translocating the metabolic intermediates between these two compartments have been characterized [M. Jackson, C. M. Stevens, L. Zhang, H. I. Zgurskaya, M. Niederweis, Chem. Rev., 10.1021/acs.chemrev.0c00869 (2020)]. In this work, we use CRISPR interference methodology in Mycobacterium smegmatis to functionally characterize an ATP-binding cassette (ABC) transporter involved in the translocation of galactan precursors across the plasma membrane. We show that genetic knockdown of the transmembrane subunit of the transporter results in severe morphological changes and the accumulation of an aberrantly long galactan precursor. Based on similarities with structures and functions of specific O-antigen ABC transporters of gram-negative bacteria [C. Whitfield, D. M. Williams, S. D. Kelly, J. Biol. Chem. 295, 10593-10609 (2020)], we propose a model for coupled synthesis and export of the galactan polymer precursor in mycobacteria.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Galactanos/metabolismo , Lipopolisacáridos/metabolismo , Mycobacterium smegmatis/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Modelos Moleculares , Mycobacterium smegmatis/genéticaRESUMEN
Xyloglucan endotransglycosylases (XETs) play key roles in the remodelling and reconstruction of plant cell walls. These enzymes catalyse homo-transglycosylation reactions with xyloglucan-derived donor and acceptor substrates and hetero-transglycosylation reactions with a variety of structurally diverse polysaccharides. In this work, we describe the basis of acceptor substrate binding specificity in non-specific Tropaeolum majus (TmXET6.3) and specific Populus tremula x tremuloides (PttXET16A) XETs, using molecular docking and molecular dynamics (MD) simulations combined with binding free energy calculations. The data indicate that the enzyme-donor (xyloglucan heptaoligosaccharide or XG-OS7)/acceptor complexes with the linear acceptors, where a backbone consisted of glucose (Glc) moieties linked via (1,4)- or (1,3)-ß-glycosidic linkages, were bound stably in the active sites of TmXET6.3 and PttXET16A. Conversely, the acceptors with the (1,6)-ß-linked Glc moieties were bound stably in TmXET6.3 but not in PttXET16A. When in the (1,4)-ß-linked Glc containing acceptors, the saccharide moieties were replaced with mannose or xylose, they bound stably in TmXET6.3 but lacked stability in PttXET16A. MD simulations of the XET-donor/acceptor complexes with acceptors derived from (1,4;1,3)-ß-glucans highlighted the importance of (1,3)-ß-glycosidic linkages and side chain positions in the acceptor substrates. Our findings explain the differences in acceptor binding specificity between non-specific and specific XETs and associate theoretical to experimental data.
Asunto(s)
Química Computacional , beta-Glucanos , Glucosa , Glicosilación , Glicosiltransferasas/metabolismo , Manosa , Simulación del Acoplamiento Molecular , Plantas/metabolismo , Polisacáridos/metabolismo , Especificidad por Sustrato , Xilanos/química , XilosaRESUMEN
We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.
Asunto(s)
Pared Celular/metabolismo , Glucanos/metabolismo , Glicosiltransferasas/metabolismo , Hordeum/metabolismo , Oligosacáridos/metabolismo , Xilanos/metabolismo , Aniones/metabolismo , Dominio Catalítico , Fluoresceínas/química , Glicosilación , Glicosiltransferasas/química , Glicosiltransferasas/genética , Hordeum/citología , Hordeum/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Familia de Multigenes , Oligosacáridos/química , Pectinas/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Especificidad por Sustrato , Ácidos Sulfónicos/químicaRESUMEN
The molecular recognition of carbohydrates by proteins plays a key role in many biological processes including immune response, pathogen entry into a cell, and cell-cell adhesion (e.g., in cancer metastasis). Carbohydrates interact with proteins mainly through hydrogen bonding, metal-ion-mediated interaction, and non-polar dispersion interactions. The role of dispersion-driven CH-π interactions (stacking) in protein-carbohydrate recognition has been underestimated for a long time considering the polar interactions to be the main forces for saccharide interactions. However, over the last few years it turns out that non-polar interactions are equally important. In this study, we analyzed the CH-π interactions employing bioinformatics (data mining, structural analysis), several experimental (isothermal titration calorimetry (ITC), X-ray crystallography), and computational techniques. The Protein Data Bank (PDB) has been used as a source of structural data. The PDB contains over 12 000 protein complexes with carbohydrates. Stacking interactions are very frequently present in such complexes (about 39 % of identified structures). The calculations and the ITC measurement results suggest that the CH-π stacking contribution to the overall binding energy ranges from 4 up to 8â kcal mol-1 . All the results show that the stacking CH-π interactions in protein-carbohydrate complexes can be considered to be a driving force of the binding in such complexes.
Asunto(s)
Carbohidratos/química , Carbono/química , Biología Computacional , Hidrógeno/química , Proteínas/química , Enlace de Hidrógeno , Técnicas In Vitro , Unión Proteica , TermodinámicaRESUMEN
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.
Asunto(s)
Pared Celular/química , Pared Celular/enzimología , Glicosiltransferasas/fisiología , Plantas/química , Plantas/enzimología , Biocatálisis , Glicosilación , Glicosiltransferasas/química , Especificidad por SustratoRESUMEN
A series of ten novel á´ -fructofuranosyl and á´ -tagatofuranosyl sulfones bearing a 1-O-phosphono moiety and three different substituents at C-2 has been prepared. Due to the structural similarities of these scaffolds to the native substrate of mycobacterial galactofuranosyltransferase GlfT2 in the transition state, we evaluated these compounds by computational methods, as well as in an enzyme assay for the possible inhibition of the mycobacterial galactan biosynthesis. Our data show that despite favorable docking scores to the active site of GlfT2, none of these compounds serve as efficient inhibitors of the enzymes involved in the mycobacterial galactan biosynthesis.
RESUMEN
KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-ß-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-ß-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-ß-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-ß-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-ß-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.
Asunto(s)
Glicosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas , Semillas/enzimología , Tropaeolum/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Germinación , Glicosilación , Glicosiltransferasas/química , Modelos Moleculares , Petroselinum/enzimología , Filogenia , Proteínas de Plantas/química , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
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 transferaseâ 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Galactosiltransferasas/metabolismo , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/enzimología , Teoría Cuántica , Proteínas Bacterianas/química , Sitios de Unión , Dominio Catalítico , Galactosa/química , Galactosa/metabolismo , Galactosiltransferasas/química , Especificidad por Sustrato , TermodinámicaRESUMEN
XynA from Erwinia chrysanthemi (EcXyn30A), belonging to glycoside hydrolase family 30 subfamily 8, is specialized for hydrolysis of 4-O-methylglucuronoxylan (GX). Carboxyl group of 4-O-methylglucuronic acid serves as a substrate recognition element interacting ionically with positively charged Arg293 of the enzyme. We determined kinetic parameters of EcXyn30A on GX, its methyl ester (GXE) and 4-O-methylglucoxylan (GXR) and compared them with behavior of the enzyme variant in which Arg293 was replaced by Ala. The modifications of the substrate carboxyl groups resulted in several thousand-fold decrease in catalytic efficiency of EcXyn30A. In contrast, the R293A replacement reduced catalytic efficiency on GX only 18-times. The main difference was in catalytic rate (kcat) which was much lower for EcXyn30A acting on the modified substrates than for the variant which exhibited similar kcat values on all three polymers. The R293A variant cleaved GX, GXE and GXR on the second glycosidic bond from branch towards the reducing end, similarly to EcXyn30A. The R293A replacement caused 15-times decrease in specific activity on MeGlcA3Xyl4, but it did not influence low activity on linear xylooligosaccharides. Docking experiments showed that MeGlcA3Xyl4 and its esterified and reduced forms were bound to both enzymes in analogous way but with different binding energies.
Asunto(s)
Endo-1,4-beta Xilanasas/metabolismo , Xilanos/química , Xilanos/metabolismo , Aspergillus niger/enzimología , Endo-1,4-beta Xilanasas/química , Hidrólisis , Cinética , Modelos Moleculares , Polimerizacion , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and ß-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.
Asunto(s)
Basidiomycota/enzimología , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , alfa-Galactosidasa/química , alfa-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Basidiomycota/clasificación , Basidiomycota/genética , Basidiomycota/crecimiento & desarrollo , Cryptococcus , Citosol/enzimología , ADN Complementario , ADN de Hongos/genética , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Genes Fúngicos/genética , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Lactosa/metabolismo , Modelos Moleculares , Peso Molecular , Conformación Proteica , Alineación de Secuencia , Análisis de Secuencia de Proteína , Especificidad por Sustrato , Temperatura , alfa-Galactosidasa/genética , alfa-Galactosidasa/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Glicosiltransferasas/química , Conformación Molecular , N-Acetilglucosaminiltransferasas/química , Polisacáridos/química , Humanos , Manosa/química , Modelos Moleculares , Simulación de Dinámica Molecular , Polisacáridos/genética , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Glicosiltransferasas/química , Glicosiltransferasas/ultraestructura , Modelos Químicos , Modelos Moleculares , Polisacáridos/química , Polisacáridos/ultraestructura , Algoritmos , Sitios de Unión , Catálisis , Simulación por Computador , Glicosilación , Cinética , Unión Proteica , Conformación ProteicaRESUMEN
KRASG12D are the most prevalent oncogenic mutations and a promising target for solid tumor therapies. However, its inhibition exhibits tremendous challenge due to the necessity of high binding affinity to obviate the need for covalent binders. Here we report the evidence of a novel class of Imidazo[1,2-a]pyridine derivative as potentially significant novel inhibitors of KRASG12D, discovered through extensive ligand-based screening against 2-[(2R)-piperidin-2-yl]-1H-indole, an important scaffold for KRASG12D inhibition via switch-I/II (S-I/II) pocket. The proposed compounds exhibited similar binding affinities and overlapped pose configurations to 2-[(2R)-piperidin-2-yl]-1H-indole, serving as a reliable starting point for drug discovery. Comparative free energy profiles demonstrated that C4 [2-methyl-3-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)imidazo[1,2-a]pyridine] effectively shifted the protein to a stable low-energy conformation via a prominent transition state. The conformational changes across the transition revealed the conformational shift of switch-I and II to a previously known off-like conformation of inactive KRASG12D with rmsd of 0.91 Å. These conformations were even more prominent than the privileged scaffold 2-[(2R)-piperidin-2-yl]-1H-indole. The representative structure overlay of C4 and another X-ray crystallography solved BI-2852 bound inactive KRASG12D revealed that Switch-I and II exhibited off-like conformations. The cumulative variance across the first eigenvalue that accounted for 57 % of the collective variance validated this on-to-off transition. In addition, the relative interaction of C4 binding showed consistent patterns with BI-2852. Taken together, our results support the inhibitory activity of [2-methyl-3-((5-phenyl-1H-1,2,4-triazol-3-yl)methyl)imidazo[1,2-a]pyridine] by shifting active KRASG12D to an inactive conformation.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras) , Piridinas , Piridinas/química , Piridinas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Humanos , Imidazoles/química , Imidazoles/farmacología , Conformación Proteica , Simulación del Acoplamiento Molecular , Unión Proteica , MutaciónRESUMEN
ß1,6-GlcNAc-transferase (C2GnT) is an important controlling factor of biological functions for many glycoproteins and its activity has been found to be altered in breast, colon, and lung cancer cells, in leukemia cells, in the lymhomonocytes of multiple sclerosis patients, leukocytes from diabetes patients, and in conditions causing an immune deficiency. The result of the action of C2GnT is the core 2 structure that is essential for the further elongation of the carbohydrate chains of O-glycans. The catalytic mechanism of this metal-ion-independent glycosyltransferase is of paramount importance and is investigated here by using quantum mechanical (QM) (density functional theory (DFT))/molecular modeling (MM) methods with different levels of theory. The structural model of the reaction site used in this report is based on the crystal structures of C2GnT. The entire enzyme-substrate system was subdivided into two different subsystems: the QM subsystem containing 206 atoms and the MM region containing 5914 atoms. Three predefined reaction coordinates were employed to investigate the catalytic mechanism. The calculated potential energy surfaces discovered the existence of a concerted SN 2-like mechanism. In this mechanism, a nucleophilic attack by O6 facilitated by proton transfer to the catalytic base and the separation of the leaving group all occur almost simultaneously. The transition state for the proposed reaction mechanism at the M06-2X/6-31G** (with diffuse functions on the O1', O5', OGlu , and O6 atoms) level was located at C1-O6=1.74â Å and C1-O1=2.86â Å. The activation energy for this mechanism was estimated to be between 20 and 29 kcal mol⻹, depending on the method used. These calculations also identified a low-barrier hydrogen bond between the nucleophile O6H and the catalytic base Glu320, and a hydrogen bond between the N-acetamino group and the glycosidic oxygen of the donor in the TS. It is proposed that these interactions contribute to a stabilization of TS and participate in the catalytic mechanism.
Asunto(s)
Biocatálisis , N-Acetilglucosaminiltransferasas/química , Humanos , Enlace de Hidrógeno , Iones/química , Metales/química , Modelos Químicos , Modelos Moleculares , N-Acetilglucosaminiltransferasas/metabolismo , Estructura Terciaria de Proteína , Teoría Cuántica , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Integrinas , Neoplasias , Humanos , Integrinas/metabolismo , Ligandos , Moléculas de Adhesión Celular , Modelos Moleculares , Neoplasias/tratamiento farmacológicoRESUMEN
In higher eukaryotes, a variety of proteins are post-translationally modified by adding O-linked N-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of O-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 O-GlcNAc transferase (uridine diphospho-N-acetylglucosamine:polypeptide ß-N-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(N)2-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 N-acetamino group of the donor participates in the catalytic mechanism.
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N-Acetilglucosaminiltransferasas/metabolismo , Teoría Cuántica , Catálisis , Modelos Moleculares , N-Acetilglucosaminiltransferasas/química , Especificidad por SustratoRESUMEN
N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated ß-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3µM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the ß-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.
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Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucógeno Fosforilasa/antagonistas & inhibidores , Urea/análogos & derivados , Animales , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Glucógeno Fosforilasa/química , Glucógeno Fosforilasa/metabolismo , Glucógeno Fosforilasa de Forma Muscular/antagonistas & inhibidores , Glucógeno Fosforilasa de Forma Muscular/química , Glucógeno Fosforilasa de Forma Muscular/metabolismo , Modelos Moleculares , Conejos , Urea/síntesis química , Urea/química , Urea/farmacologíaRESUMEN
Cholera is a life-threatening diarrhoeal disease caused by ingestion of Vibrio cholerae. There are at least 200 serogroups of V. cholerae but only two of them are causing epidemics - O1 and O139 serogroups. Fragmentation analysis of O-antigen, also known as O-specific polysaccharide (OSP), from lipopolysaccharide (LPS) is important to obtain new information about its structure, such as fragmentation patterns and fragment structures. In the present study, OSP and core (OSPc) structure from V. cholerae O139 was studied using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) and direct injection electrospray ionization (ESI)-MS methods. MALDI-TOF analysis was performed in positive-ion reflectron mode, while ESI-MS was performed in negative ionization mode. ESI-MS analysis was followed by ESI-MS/MS analysis. Using this analytical approach, we managed to obtain two possible fragmentation pathways of OSP from V. cholerae O139. Mutual sign of these two pathways is shortening the length of the oligosaccharide by neutral loss of monosaccharide residues. Additionally, liquid chromatography-MS analysis was performed to separate depicted molecular forms of OSPc.
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Vibrio cholerae O139 , Vibrio cholerae , Cromatografía Liquida , Antígenos O , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem , Vibrio cholerae/químicaRESUMEN
Here, we present a comparative structure-function study of a nematode and a plant core α1,3-fucosyltransferase based on deletion and point mutations of the coding regions of Caenorhabditis elegans FUT-1 and Arabidopsis thaliana FucTA (FUT11). In particular, our results reveal a novel "first cluster motif" shared by both core and Lewis-type α1,3-fucosyltransferases of the GT10 family. To evaluate the role of the conserved serine within this motif, this residue was replaced with alanine in FucTA (S218) and FUT-1 (S243). The S218A replacement completely abolished the enzyme activity of FucTA, while the S243A mutant of FUT-1 retained 20% of the "wild-type" activity. Based on the results of homology modeling of FucTA, other residues potentially involved in the donor substrate binding were examined, and mutations of N219 and R226 dramatically affected enzymatic activity. Finally, as both FucTA and FUT-1 were shown to be N-glycosylated, we examined the putative N-glycosylation sites. While alanine replacements at single potential N-glycosylation sites of FucTA resulted in a loss of up to 80% of the activity, a triple glycosylation site mutant still retained 5%, as compared to the control. In summary, our data indicate similar trends in structure-function relationships of distantly related enzymes which perform similar biochemical reactions and form the basis for future work aimed at understanding the structure of α1,3-fucosyltransferases in general.