RESUMEN
Differential scanning fluorimetry (DSF) is a technique that reports protein thermal stability via the selective recognition of unfolded states by fluorogenic dyes. However, DSF applications remain limited by protein incompatibilities with existing DSF dyes. Here we overcome this obstacle with the development of a protein-adaptive DSF platform (paDSF) that combines a dye library 'Aurora' with a streamlined procedure to identify protein-dye pairs on demand. paDSF was successfully applied to 94% (66 of 70) of proteins, tripling the previous compatibility and delivering assays for 66 functionally and biochemically diverse proteins, including 10 from severe acute respiratory syndrome coronavirus 2. We find that paDSF can be used to monitor biological processes that were previously inaccessible, demonstrated for the interdomain allostery of O-GlcNAc transferase. The chemical diversity and varied selectivities of Aurora dyes suggest that paDSF functionality may be readily extended. paDSF is a generalizable tool to interrogate protein stability, dynamics and ligand binding.
RESUMEN
Human O-linked ß-N-acetylglucosaminidase (hOGA) is one of the two enzymes involved in nuclear and cytoplasmic protein O-GlcNAcylation, an essential post-translational modification. The enzyme catalyzes the hydrolysis of the GlcNAc-O-(Ser/Thr) glycosidic bonds via anchimeric assistance through the 2-acetamido group of the GlcNAc sugar. However, the conformational itinerary of the GlcNAc ring during catalysis remains unclear. Here we report the crystal structure of wild type hOGA in complex with a nonhydrolyzable glycopeptide substrate and elucidate the full enzyme catalytic mechanism using QM/MM metadynamics. We show that the enzyme can bind the substrate in either a chair- or a boat-like conformation, but only the latter is catalytically competent, leading to the reaction products via 1,4B/1S3 â [4E] â 4C1 and 4C1 â [4E] â 1,4B/1S3 conformational itineraries for the first and second catalytic reaction steps, respectively. Our results reconcile previous experimental observations for human and bacterial OGA and will aid the development of more effective OGA inhibitors for diseases associated with impaired O-GlcNAcylation.
RESUMEN
The modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulator of cell physiology. O-GlcNAc is installed on over a thousand proteins by just one enzyme, O-GlcNAc transferase (OGT). How OGT is regulated is therefore a topic of interest. To gain insight into these questions, we used OGT to perform phage display selection from an unbiased library of ~109 peptides of 15 amino acids in length. Following rounds of selection and deep mutational panning, we identified a high-fidelity peptide consensus sequence, [Y/F]-x-P-x-Y-x-[I/M/F], that drives peptide binding to OGT. Peptides containing this sequence bind to OGT in the high nanomolar to low micromolar range and inhibit OGT in a noncompetitive manner with low micromolar potencies. X-ray structural analyses of OGT in complex with a peptide containing this motif surprisingly revealed binding to an exosite proximal to the active site of OGT. This structure defines the detailed molecular basis driving peptide binding and explains the need for specific residues within the sequence motif. Analysis of the human proteome revealed this motif within 52 nuclear and cytoplasmic proteins. Collectively, these data suggest a mode of regulation of OGT by which polypeptides can bind to this exosite to cause allosteric inhibition of OGT through steric occlusion of its active site. We expect that these insights will drive improved understanding of the regulation of OGT within cells and enable the development of new chemical tools to exert fine control over OGT activity.
Asunto(s)
Bacteriófagos , Péptidos , Humanos , Secuencia de Aminoácidos , N-Acetilglucosaminiltransferasas/metabolismo , Mutación , Bacteriófagos/metabolismoRESUMEN
The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein.
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Proteína Huntingtina , Proteínas Nucleares , Microscopía por Crioelectrón , Proteína Huntingtina/química , Proteínas Nucleares/química , HumanosRESUMEN
Flexible in vitro methods alter the course of biological discoveries. Differential Scanning Fluorimetry (DSF) is a particularly versatile technique which reports protein thermal unfolding via fluorogenic dye. However, applications of DSF are limited by widespread protein incompatibilities with the available DSF dyes. Here, we enable DSF applications for 66 of 70 tested proteins (94%) including 10 from the SARS-CoV2 virus using a chemically diverse dye library, Aurora, to identify compatible dye-protein pairs in high throughput. We find that this protein-adaptive DSF platform (paDSF) not only triples the previous protein compatibility, but also fundamentally extends the processes observable by DSF, including interdomain allostery in O-GlcNAc Transferase (OGT). paDSF enables routine measurement of protein stability, dynamics, and ligand binding.
RESUMEN
Glycosyltransferases are a superfamily of enzymes that are notoriously difficult to inhibit. Here we apply an mRNA display technology integrated with genetic code reprogramming, referred to as the RaPID (random non-standard peptides integrated discovery) system, to identify macrocyclic peptides with high binding affinities for O-GlcNAc transferase (OGT). These macrocycles inhibit OGT activity through an allosteric mechanism that is driven by their binding to the tetratricopeptide repeats of OGT. Saturation mutagenesis in a maturation screen using 39 amino acids, including 22 non-canonical residues, led to an improved unnatural macrocycle that is ≈40â times more potent than the parent compound (Ki app =1.5â nM). Subsequent derivatization delivered a biotinylated derivative that enabled one-step affinity purification of OGT from complex samples. The high potency and novel mechanism of action of these OGT ligands should enable new approaches to elucidate the specificity and regulation of OGT.
Asunto(s)
N-Acetilglucosaminiltransferasas , Péptidos , Péptidos/genética , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , MutagénesisRESUMEN
Anticancer drug response is determined by genetic and epigenetic mechanisms. To identify the epigenetic regulators of anticancer drug response, we conducted a chemical epigenetic screen using chemical probes that target different epigenetic modulators. In this screen, we tested 31 epigenetic probes in combination with 14 mechanistically diverse anticancer agents and identified 8 epigenetic probes that significantly potentiate the cytotoxicity of TAK-243, a first-in-class ubiquitin-activating enzyme (UBA1) inhibitor evaluated in several solid and hematologic malignancies. These probes are TP-472, GSK864, A-196, UNC1999, SGC-CBP30, and PFI-4 (and its related analogues GSK6853 and GSK5959), and they target BRD9/7, mutant IDH1, SUV420H1/2, EZH2/1, p300/CBP, and BRPF1B, respectively. In contrast to epigenetic probes, negative control compounds did not have a significant impact on TAK-243 cytotoxicity. Potentiation of TAK-243 cytotoxicity was associated with reduced ubiquitylation and induction of apoptosis. Mechanistically, these epigenetic probes exerted their potentiation by inhibiting the efflux transporter ATP-binding cassette subfamily G member 2 (ABCG2) without inducing significant changes in the ubiquitylation pathways or ABCG2 expression levels. As assessed by docking analysis, the identified probes could potentially interact with ABCG2. Based on these data, we have developed a cell-based assay that can quantitatively evaluate ABCG2 inhibition by drug candidates. In conclusion, our study identifies epigenetic probes that profoundly potentiate TAK-243 cytotoxicity through off-target ABCG2 inhibition. We also provide experimental evidence that several negative control compounds cannot exclude a subset of off-target effects of chemical probes. Finally, potentiation of TAK-243 cytotoxicity can serve as a quantitative measure of ABCG2-inhibitory activity.
Asunto(s)
Antineoplásicos , Resistencia a Antineoplásicos , Enzimas Activadoras de Ubiquitina , Antineoplásicos/farmacología , Antineoplásicos/química , Epigénesis Genética , Adenosina TrifosfatoRESUMEN
O-GlcNAcylation is an essential post-translational modification installed by the enzyme O-ß-N-acetyl-d-glucosaminyl transferase (OGT). Modulating this enzyme would be extremely valuable to better understand its role in the development of serious human pathologies, such as diabetes and cancer. However, the limited availability of potent and selective inhibitors hinders the validation of this potential therapeutic target. To explore new chemotypes that target the active site of OGT, we performed virtual screening of a large library of commercially available compounds with drug-like properties. We purchased samples of the most promising virtual hits and used enzyme assays to identify authentic leads. Structure-activity relationships of the best identified OGT inhibitor were explored by generating a small library of derivatives. Our best hit displays a novel uridine mimetic scaffold and inhibited the recombinant enzyme with an IC50 value of 7 µM. The current hit represents an excellent starting point for designing and developing a new set of OGT inhibitors that may prove useful for exploring the biology of OGT.
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N-Acetilglucosaminiltransferasas , Procesamiento Proteico-Postraduccional , Humanos , N-Acetilglucosaminiltransferasas/metabolismo , Investigación , Relación Estructura-ActividadRESUMEN
The O-linked ß-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.
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Aminoácidos/metabolismo , Cromo/metabolismo , Microscopía por Crioelectrón/métodos , Ácidos Nicotínicos/metabolismo , Aminoácidos/química , Cromo/química , Cristalografía por Rayos X , Glicómica , Mutación/genética , Ácidos Nicotínicos/química , Estructura Secundaria de ProteínaRESUMEN
O-linked N-acetylglucosamine (O-GlcNAc) is protein modification that is emerging as a regulator of diverse aspects of cellular physiology. Aberrant O-GlcNAcylation has been linked to several diseases, spurring the creation of methods to detect and perturb the activity of the two enzymes that govern this modification - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Here we summarize assays used for these two enzymes. We also detail the latest structure-guided development of inhibitors of these two enzymes and touch on selected reports that underscore the utility of inhibitors as tools for uncovering the diverse roles of O-GlcNAc in cell function. Finally, we summarize recent reports on the potential therapeutic benefits of antagonizing these enzymes and comment on outstanding challenges within the field.
Asunto(s)
Acetilglucosamina , N-Acetilglucosaminiltransferasas , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator ß-catenin in its O-GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of ß-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the O-GlcNAcylation status of ß-catenin in HeLa cells. The changes in O-GlcNAcylation of ß-catenin were varied by perturbing global cellular O-GlcNAc levels with the inhibitors of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.
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Acetilglucosamina/metabolismo , Ingeniería Metabólica , Imagen Óptica , beta Catenina/metabolismo , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Glicosilación , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Oligosacáridos/química , Oligosacáridos/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Despite its central importance as a regulator of cellular physiology, identification and precise mapping of O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification (PTM) sites in proteins by mass spectrometry (MS) remains a considerable technical challenge. This is due in part to cleavage of the glycosidic bond occurring prior to the peptide backbone during collisionally activated dissociation (CAD), which leads to generation of characteristic oxocarbenium ions and impairs glycosite localization. Herein, we leverage CAD-induced oxocarbenium ion generation to trigger ultraviolet photodissociation (UVPD), an alternate high-energy deposition method that offers extensive fragmentation of peptides while leaving the glycosite intact. Upon activation using UV laser pulses, efficient photodissociation of glycopeptides is achieved with production of multiple sequence ions that enable robust and precise localization of O-GlcNAc sites. Application of this method to tryptic peptides originating from O-GlcNAcylated proteins TAB1 and Polyhomeotic confirmed previously reported O-GlcNAc sites in TAB1 (S395 and S396) and uncovered new sites within both proteins. We expect this strategy will complement existing MS/MS methods and be broadly useful for mapping O-GlcNAcylated residues of both proteins and proteomes.
Asunto(s)
Acetilglucosamina/química , Proteínas/química , Acetilglucosamina/metabolismo , Animales , Perros , Espectrometría de Masas , Procesos Fotoquímicos , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Rayos UltravioletaRESUMEN
Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high-throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead-based strategy to measure the group-transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O-GlcNAc transferase (OGT) as a model system through detailed Michaelis-Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high-throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group-transfer enzymes.
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Pruebas de Enzimas/métodos , N-Acetilglucosaminiltransferasas/metabolismo , Espectrometría de Fluorescencia/métodos , Glicosilación , Cinética , Especificidad por SustratoRESUMEN
An unstudied ß-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and a robust thermal stability at 50 °C within 24 h. The addition of urea (1 M) or sodium dodecyl sulfate (1% w/v) reduced the activity of the enzyme by 44% and 58%, respectively, whereas the addition of divalent metal ions had no effect on the enzymatic activity. PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) strongly inhibited the enzyme in sub-micromolar concentrations. The ß-N-acetylhexosaminidase was able to hydrolyze ß1,2-linked, ß1,3-linked, ß1,4-linked, and ß1,6-linked GlcNAc residues from the non-reducing end of various tested glycan standards, including bisecting GlcNAc from one of the tested hybrid-type N-glycan substrates. A mutational study revealed that the amino acids D306 and E307 bear the catalytically relevant side acid/base side chains. When coupled with a chitinase, the ß-N-acetylhexosaminidase was able to generate GlcNAc directly from colloidal chitin, which showed the potential of this enzyme for biotechnological applications.
Asunto(s)
Actinomycetales/metabolismo , Disacáridos/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Aminoácidos/metabolismo , Quitina/metabolismo , Quitinasas/metabolismo , Escherichia coli/metabolismo , Oximas/metabolismo , Fenilcarbamatos/metabolismo , Microbiología del SueloRESUMEN
The modification of proteins with O-linked N-acetylglucosamine ( O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells have proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here, we report on the creation of fluorescent uridine 5'-diphospho- N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT, we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time- and dose-dependent manner. Because no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-color pulse chase experiments and monitoring OGT activity on specific protein substrates in live cells.
Asunto(s)
Acetilglucosamina/química , Fluorescencia , N-Acetilglucosaminiltransferasas/química , Acetilglucosamina/metabolismo , Glicosilación , Células HeLa , Humanos , Estructura Molecular , N-Acetilglucosaminiltransferasas/metabolismoRESUMEN
O-GlcNAc transferase (OGT) catalyzes the installation of N-acetylglucosamine (GlcNAc) O-linked to nucleocytoplasmic proteins (O-GlcNAc) within multicellular eukaryotes. OGT shows surprising tolerance for structural changes in the sugar component of its nucleotide sugar donor substrate, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Here, we find that OGT uses UDP-glucose to install O-linked glucose (O-Glc) onto proteins only 25-fold less efficiently than O-GlcNAc. Spurred by this observation, we show that OGT transfers 2-azido-2-deoxy-d-glucose (GlcAz) in vitro from UDP-GlcAz to proteins. Further, feeding cells with per-O-acetyl GlcAz (AcGlcAz), in combination with inhibition or inducible knockout of OGT, shows OGT-dependent modification of nuclear and cytoplasmic proteins with O-GlcAz as detected using microscopy, immunoblot, and proteomics. We find that O-GlcAz is reversible within cells, and an unidentified cellular enzyme exists to cleave O-Glc that can also process O-GlcAz. We anticipate that AcGlcAz will prove to be a useful tool to study the O-GlcNAc modification. We also speculate that, given the high concentration of UDP-Glc within certain mammalian tissues, O-Glc may exist within mammals and serve as a physiologically relevant modification.
Asunto(s)
Azidas/química , Desoxiglucosa/análogos & derivados , Glucosa/química , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Azidas/metabolismo , Células COS , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Chlorocebus aethiops , Desoxiglucosa/química , Glucosa/análogos & derivados , Glucosa/metabolismo , Glicosilación , Humanos , Glicoproteínas de Membrana/metabolismo , Ingeniería Metabólica , Ratones , N-Acetilglucosaminiltransferasas/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Especificidad por Sustrato , Tritio , Uridina Difosfato Glucosa/análogos & derivados , Uridina Difosfato Glucosa/química , Uridina Difosfato Glucosa/metabolismo , beta-N-Acetilhexosaminidasas/química , Proteínas tau/metabolismoRESUMEN
Mammalian ß-hexosaminidases have been shown to play essential roles in cellular physiology and health. These enzymes are responsible for the cleavage of the monosaccharides N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) from cellular substrates. One of these ß-hexosaminidases, hexosaminidase D (HexD), encoded by the HEXDC gene, has received little attention. No mechanistic studies have focused on the role of this unusual nucleocytoplasmically localized ß-hexosaminidase, and its cellular function remains unknown. Using a series of kinetic and mechanistic investigations into HexD, we define the precise catalytic mechanism of this enzyme and establish the identities of key enzymic residues. The preparation of synthetic aryl N-acetylgalactosaminide substrates for HexD in combination with measurements of kinetic parameters for wild-type and mutant enzymes, linear free energy analyses of the enzyme-catalyzed hydrolysis of these substrates, evaluation of the reaction by nuclear magnetic resonance, and inhibition studies collectively reveal the detailed mechanism of action employed by HexD. HexD is a retaining glycosidase that operates using a substrate-assisted catalytic mechanism, has a preference for galactosaminide over glucosaminide substrates, and shows a pH optimum in its second-order rate constant at pH 6.5-7.0. The catalytically important residues are Asp148 and Glu149, with Glu149 serving as the general acid/base residue and Asp148 as the polarizing residue. HexD is inhibited by Gal-NAG-thiazoline (Ki = 420 nM). The fundamental insights gained from this study will aid in the development of potent and selective probes for HexD, which will serve as useful tools to improve our understanding of the physiological role played by this unusual enzyme.
Asunto(s)
Inhibidores Enzimáticos/química , Hexosaminidasas/antagonistas & inhibidores , Hexosaminidasas/química , Tiazolidinas/química , Catálisis , Hexosaminidasas/genética , Hexosaminidasas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , CinéticaRESUMEN
Clostridium perfringens is an opportunistic pathogen of humans and animals whose genome encodes a wide variety of putative carbohydrate-hydrolyzing enzymes that are increasingly being shown to be directed toward the cleavage of host glycans. Among these putative enzymes is a member of glycoside hydrolase family 123. Here we show that the recombinant enzyme (referred to as CpNga123) encoded by the gene cloned from C. perfringens strain ATCC 13124 (locus tag CPF_1473) is a ß-N-acetylgalactosaminidase, similar to NgaP from Paenibacillus sp. TS12. Like NgaP, CpNga123 was able to cleave the terminal ß-D-GalNAc-(1â4)-D-Gal and ß-D-GalNAc-(1â3)-D-Gal motifs that would be found in glycosphigolipids. The X-ray crystal structure of CpNga123 revealed it to have an N-terminal ß-sandwich domain and a (ß/α)8-barrel catalytic domain with a C-terminal α-helical elaboration. The structures determined in complex with reaction products provide details of the -1 subsite architecture, catalytic residues, and a structural change in the active site that is likely required to enable hydrolysis of the glycosidic bond by promoting engagement of the substrate by the catalytic residues. The features of the active site support the likelihood of a substrate-assisted catalytic mechanism for this enzyme. The structures of an inactive mutant of CpNga123 in complex with intact GA2 and Gb4 glycosphingolipid motifs reveal insight into aglycon recognition and suggest that the kinked or pleated conformation of GA2 caused by the ß-1,4-linkage between N-acetylgalactosamine and galactose, and the accommodation of this conformation by the enzyme active site, may be responsible for greater activity on GA2.
Asunto(s)
Clostridium perfringens/metabolismo , Glucolípidos/metabolismo , Glicósido Hidrolasas/metabolismo , Catálisis , Dominio Catalítico/fisiología , Galactosa/metabolismo , Hidrólisis , beta-N-Acetil-Galactosaminidasa/metabolismoRESUMEN
Low-molecular-weight ice recrystallization inhibitors (IRIs) are ideal cryoprotectants that control the growth of ice and mitigate cell damage during freezing. Herein, we describe a detailed study correlating the ice recrystallization inhibition activity and the cryopreservation ability with the structure of O-aryl-glycosides. Many effective IRIs are efficient cryoadditives for the freezing of red blood cells (RBCs). One effective cryoadditive did not inhibit ice recrystallization but instead inhibited ice nucleation, demonstrating the significance of inhibiting both processes and illustrating the importance of this emerging class of cryoprotectants.