RESUMEN
The vast majority of glycosidases characterized to date follow one of the variations of the 'Koshland' mechanisms1 to hydrolyse glycosidic bonds through substitution reactions. Here we describe a large-scale screen of a human gut microbiome metagenomic library using an assay that selectively identifies non-Koshland glycosidase activities2. Using this, we identify a cluster of enzymes with extremely broad substrate specificities and thoroughly characterize these, mechanistically and structurally. These enzymes not only break glycosidic linkages of both α and ß stereochemistry and multiple connectivities, but also cleave substrates that are not hydrolysed by standard glycosidases. These include thioglycosides, such as the glucosinolates from plants, and pseudoglycosidic bonds of pharmaceuticals such as acarbose. This is achieved through a distinct mechanism of hydrolysis that involves oxidation/reduction and elimination/hydration steps, each catalysed by enzyme modules that are in many cases interchangeable between organisms and substrate classes. Homologues of these enzymes occur in both Gram-positive and Gram-negative bacteria associated with the gut microbiome and other body parts, as well as other environments, such as soil and sea. Such alternative step-wise mechanisms appear to constitute largely unrecognized but abundant pathways for glycan degradation as part of the metabolism of carbohydrates in bacteria.
Asunto(s)
Bacterias , Microbioma Gastrointestinal , Glicósido Hidrolasas , Polisacáridos , Humanos , Acarbosa/química , Acarbosa/metabolismo , Bacterias/enzimología , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Biocatálisis , Glucosinolatos/metabolismo , Glucosinolatos/química , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/química , Hidrólisis , Metagenoma , Oxidación-Reducción , Plantas/química , Polisacáridos/metabolismo , Polisacáridos/química , Agua de Mar/microbiología , Microbiología del Suelo , Especificidad por Sustrato , MasculinoRESUMEN
Mucin-type O-glycosylation is a post-translational modification present at the interface between cells where it has important roles in cellular communication. However, deciphering the function of O-glycoproteins and O-glycans can be challenging, especially as few enzymes are available for their assembly or selective degradation. Here, to address this deficiency, we developed a genetically encoded screening methodology for the discovery and engineering of the diverse classes of enzymes that act on O-glycoproteins. The method uses Escherichia coli that have been engineered to produce an O-glycosylated fluorescence resonance energy transfer probe that can be used to screen for O-glycopeptidase activity. Subsequent cleavage of the substrate by O-glycopeptidases provides a read-out of the glycosylation state of the probe, allowing the method to also be used to assay glycosidases and glycosyltransferases. We further show the potential of this methodology in the first ultrahigh-throughput-directed evolution of an O-glycopeptidase.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Mucinas , Mucinas/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Glicoproteínas/química , Glicosilación , Polisacáridos/químicaRESUMEN
Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions. Terminal glycan structures include Lewis antigens synthesized by a collection of α1,3/4-fucosyltransferases (CAZy GT10 family). At present, the only available crystallographic structure of a GT10 member is that of the Helicobacter pylori α1,3-fucosyltransferase, but mammalian GT10 fucosyltransferases are distinct in sequence and substrate specificity compared with the bacterial enzyme. Here, we determined crystal structures of human FUT9, an α1,3-fucosyltransferase that generates Lewisx and Lewisy antigens, in complex with GDP, acceptor glycans, and as a FUT9-donor analog-acceptor Michaelis complex. The structures reveal substrate specificity determinants and allow prediction of a catalytic model supported by kinetic analyses of numerous active site mutants. Comparisons with other GT10 fucosyltransferases and GT-B fold glycosyltransferases provide evidence for modular evolution of donor- and acceptor-binding sites and specificity for Lewis antigen synthesis among mammalian GT10 fucosyltransferases.
Asunto(s)
Fucosiltransferasas , Glicosiltransferasas , Animales , Humanos , Fucosiltransferasas/genética , Fucosiltransferasas/química , Fucosiltransferasas/metabolismo , Antígenos del Grupo Sanguíneo de Lewis , Polisacáridos/metabolismo , MamíferosRESUMEN
Influenza A viruses and the bacterium Streptococcus pneumoniae (pneumococci) both express neuraminidases that catalyze release of sialic acid residues from oligosaccharides and glycoproteins. Although these respiratory pathogen neuraminidases function in a similar environment, it remains unclear if these enzymes use similar mechanisms for sialic acid cleavage. Here, we compared the enzymatic properties of neuraminidases from two influenza A subtypes (N1 and N2) and the pneumococcal strain TIGR4 (NanA, NanB, and NanC). Insect cell-produced N1 and N2 tetramers exhibited calcium-dependent activities and stabilities that varied with pH. In contrast, E. coli-produced NanA, NanB, and NanC were isolated as calcium insensitive monomers with stabilities that were more resistant to pH changes. Using a synthetic substrate (MUNANA), all neuraminidases showed similar pH optimums (pH 6-7) that were primarily defined by changes in catalytic rate rather than substrate binding affinity. Upon using a multivalent substrate (fetuin sialoglycans), much higher specific activities were observed for pneumococcal neuraminidases that contain an additional lectin domain. In virions, N1 and especially N2 also showed enhanced specific activity toward fetuin that was lost upon the addition of detergent, indicating the sialic acid-binding capacity of neighboring hemagglutinin molecules likely contributes to catalysis of natural multivalent substrates. These results demonstrate that influenza and pneumococcal neuraminidases have evolved similar yet distinct strategies to optimize their catalytic activity.
Asunto(s)
Virus de la Influenza A , Ácido N-Acetilneuramínico , Neuraminidasa , Calcio/metabolismo , Catálisis , Escherichia coli/enzimología , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismo , Streptococcus pneumoniae/enzimología , Virus de la Influenza A/enzimología , Animales , Línea CelularRESUMEN
Sulfation is widespread in nature and plays an important role in modulating biological function. Among the strategies developed by microbes to access sulfated oligosaccharides as a nutrient source is the production of 6-sulfoGlcNAcases to selectively release 6-sulfoGlcNAc from target oligosaccharides. Thus far, all 6-sulfoGlcNAcases identified have belonged to the large GH20 family of ß-hexosaminidases. Ηere, we identify and characterize a new, highly specific non-GH20 6-sulfoGlcNAcase from Streptococcus pneumoniae TIGR4, Sp_0475 with a greater than 110,000-fold preference toward N-acetyl-ß-D-glucosamine-6-sulfate substrates over the nonsulfated version. Sp_0475 shares distant sequence homology with enzymes of GH20 and with the newly formed GH163 family. However, the sequence similarity between them is sufficiently low that Sp_0475 has been assigned as the founding member of a new glycoside hydrolase family, GH185. By combining results from site-directed mutagenesis with mechanistic studies and bioinformatics we provide insight into the substrate specificity, mechanism, and key active site residues of Sp_0475. Enzymes of the GH185 family follow a substrate-assisted mechanism, consistent with their distant homology to the GH20 family, but the catalytic residues involved are quite different. Taken together, our results highlight in more detail how microbes can degrade sulfated oligosaccharides for nutrients.
RESUMEN
Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near - but not in - the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model ß-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.
Asunto(s)
Carbohidratos , Glicósido Hidrolasas , Glicósido Hidrolasas/metabolismo , Prueba de Estudio Conceptual , LigandosRESUMEN
Mimics of the complex flavonol glycoside montbretin A in which a flavonol moiety is coupled to a caffeic acid via partially peptidic linkers have proved to be potent inhibitors of human pancreatic alpha-amylase with potential as therapeutics for control of blood glucose levels. After exploring optimal linker length, a synthetic route to a version with a branched linker was devised based on the structure of the enzyme/inhibitor complex. The resultant branched inhibitors were shown to retain nanomolar potency even when decorated with polymers as a means of modifying solubility. Similar improvements, along with nanomolar affinity, could also be achieved through conjugation to cyclodextrins which have the potential to bind to starch binding sites found on the surface of human amylase. Incorporation of a conjugatable branch into this unusual pharmacophore thereby affords considerable flexibility for further modifications to improve pharmacokinetic behaviour or as a site for attachment of capture tags or fluorophores.
RESUMEN
Suitably configured allyl ethers of unsaturated cyclitols act as substrates of ß-glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of ß-glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter-intuitive trend wherein the most electronegative substituents yielded the most labile pseudo-glycosidic linkages. Structures of complexes with the Sulfolobus ß-glucosidase revealed similar enzyme-ligand interactions to those seen in complexes with a 2-fluorosugar inhibitor, the lone exception being displacement of tyrosine 322 from the active site by the halogen. Mutation of Y322 to Y322F largely abolished glycosidase activity, consistent with lost interactions at O5, but minimally affected (7-fold) rates of carbasugar hydrolysis, yielding a more selective enzyme for unsaturated cyclitol ether hydrolysis.
Asunto(s)
Ciclitoles , Ciclitoles/química , Glicósido Hidrolasas/metabolismo , Glicósidos , Dominio Catalítico , Inhibidores Enzimáticos/farmacologíaRESUMEN
The prospect of producing human-like glycoproteins in bacteria is becoming attractive as an alternative to already-established but costly mammalian cell expression systems. We previously described an Escherichia coli expression platform that uses a dual-plasmid approach to produce simple mucin type O-glycoproteins: one plasmid encoding the target protein and another O-glycosylation machinery. Here, we expand the capabilities of our platform to carry out sialylation and demonstrate the high-yielding production of human interferon α2b and human growth hormone bearing mono- and disialylated T-antigen glycans. This is achieved through engineering an E. coli strain to produce CMP-Neu5Ac and introducing various α-2,3- and α-2,6 mammalian or bacterial sialyltransferases into our O-glycosylation operons. We further demonstrate that mammalian sialyltransferases, including porcine ST3Gal1, human ST6GalNAc2 and human ST6GalNAc4, are very effective in vivo and outperform some of the bacterial sialyltransferases tested, including Campylobacter jejuni Cst-I and Cst-II. In the process, we came upon a way of modifying T-Antigen with Kdo, using a previously uncharacterised Kdo-transferase activity of porcine ST3Gal1. Ultimately, the heterologous expression of mammalian sialyltransferases in E. coli shows promise for the further development of bacterial systems in therapeutic glycoprotein production.
Asunto(s)
Escherichia coli , Sialiltransferasas , Animales , Antígenos Virales de Tumores , Escherichia coli/genética , Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Mamíferos/metabolismo , Mucinas/genética , Mucinas/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , PorcinosRESUMEN
Propionibacterium acnes, though generally considered part of the normal flora of human skin, is an opportunistic pathogen associated with acne vulgaris as well as other diseases, including endocarditis, endophthalmitis and prosthetic joint infections. Its virulence potential is also supported by knowledge gained from its sequenced genome. Indeed, a vaccine targeting a putative cell wall-anchored P. acnes sialidase has been shown to suppress cytotoxicity and pro-inflammatory cytokine release induced by the organism, and is proposed as an alternative treatment for P. acnes-associated diseases. Here, we report the crystal structures of the surface sialidase and its complex with the transition-state mimic Neu5Ac2en. Our structural and kinetic analyses, together with insight from a glycan array screen, which probes subtle specificities of the sialidase for α-2,3-sialosides, provide a basis for the structure-based design of novel small-molecule therapeutics against P. acnes infections.
Asunto(s)
Acné Vulgar , Propionibacterium acnes , Acné Vulgar/tratamiento farmacológico , Acné Vulgar/microbiología , Humanos , Neuraminidasa , PielRESUMEN
Supplementing influenza vaccines with recombinant neuraminidase (rNA) antigens remains a promising approach for improving suboptimal vaccine efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced by the baculovirus/insect cell system. The rNAs were designed with different tetramerization motifs and NA domains from a recent H1N1 vaccine strain (A/Brisbane/02/2018) and compared for enzymatic properties, antigenicity, stability, and protection in mice. We found that the enzymatic properties differ between rNAs containing the NA head domain versus the full ectodomain, the formation of higher-order rNA oligomers is tetramerization domain dependent, whereas the protective efficacy is more contingent on the combination of the tetramerization and NA domains. Following single-dose immunizations, an rNA possessing the full ectodomain and the tetramerization motif from the human vasodilator-stimulated phosphoprotein provided much better protection than an rNA with â¼10-fold more enzymatically active molecules that is comprised of the head domain and the same tetramerization motif. In contrast, these two rNA designs provided comparable protection when the tetramerization motif from the tetrabrachion protein was used instead. These findings demonstrate that individual rNAs should be thoroughly evaluated for vaccine development, as the heterologous domain combination can result in rNAs with similar key attributes that vastly differ in protection. IMPORTANCE For several decades, it has been proposed that influenza vaccines could be supplemented with recombinant neuraminidase (rNA) to improve efficacy. However, some key questions for manufacturing stable and immunogenic rNAs remain to be answered. We show here that the tetramerization motifs and NA domains included in the rNA construct design can have a profound impact on the biochemical, immunogenic, and protective properties. We also show that the single-dose immunization regimen is more informative for assessing the rNA immune response and protective efficacy, which is surprisingly more dependent on the specific combination of NA and tetramerization domains than common attributes for evaluating NA. Our findings may help to optimize the design of rNAs that can be used to improve or develop influenza vaccines.
Asunto(s)
Anticuerpos Antivirales/sangre , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H1N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/inmunología , Neuraminidasa/genética , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Antivirales/inmunología , Baculoviridae/genética , Baculoviridae/metabolismo , Protección Cruzada , Femenino , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Ratones , Ratones Endogámicos DBA , Neuraminidasa/inmunología , Vacunación , Desarrollo de Vacunas , Eficacia de las VacunasRESUMEN
Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. Here, in crystallo enzymology with the catalytically active bacterial cellulose synthase BcsA-BcsB complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate- and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a 'finger helix' that contacts the polymer's terminal glucose. Cooperating with BcsA's gating loop, the finger helix moves 'up' and 'down' in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA's transmembrane channel. This mechanism is validated experimentally by tethering BcsA's finger helix, which inhibits polymer translocation but not elongation.
Asunto(s)
Celulosa/biosíntesis , Celulosa/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Membranas Intracelulares/metabolismo , Celulosa/química , Cristalografía por Rayos X , Glucosa/metabolismo , Membranas Intracelulares/química , Modelos Moleculares , Movimiento , Estructura Secundaria de Proteína , Proteolípidos/química , Proteolípidos/metabolismo , Rhodobacter sphaeroides/enzimología , Especificidad por SustratoRESUMEN
Gaucher disease is a lysosomal storage disorder caused by mutations which destabilize the native folded form of GCase, triggering degradation and ultimately resulting in low enzyme activity. Pharmacological chaperones (PCs) which stabilize mutant GCase have been used to increase lysosomal activity through improving trafficking efficiency. By engineering their inherent basicity, we have synthesized PCs that change conformation between the ER and the lysosomal environment, thus weakening binding to GCase after its successful trafficking to the lysosome. NMR studies confirmed the conformational change while X-ray data reveal bound conformations and binding modes. These results were further corroborated by cell studies showing increases in GCase activity when using the pH-switchable probe at low dosing. Preliminary in vivo assays with humanized mouse models of Gaucher showed enhanced GCase activity levels in relevant tissues, including the brain, further supporting their potential.
Asunto(s)
Enfermedad de Gaucher , Glucosilceramidasa , Animales , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/genética , Glucosilceramidasa/química , Concentración de Iones de Hidrógeno , Ratones , Modelos Animales , Chaperonas Moleculares/química , MutaciónRESUMEN
Transfusion of blood, or more commonly red blood cells (RBCs), is integral to health care systems worldwide but requires careful matching of blood types to avoid serious adverse consequences. Of the four main blood types, A, B, AB, and O, only O can be given to any patient. This universal donor O-type blood is crucial for emergency situations where time or resources for typing are limited, so it is often in short supply. A and B blood differ from the O type in the presence of an additional sugar antigen (GalNAc and Gal, respectively) on the core H-antigen found on O-type RBCs. Thus, conversion of A, B, and AB RBCs to O-type RBCs should be achievable by removal of that sugar with an appropriate glycosidase. The first demonstration of a B-to-O conversion by Goldstein in 1982 required massive amounts of enzyme but enabled proof-of-principle transfusions without adverse effects in humans. New α-galactosidases and α-N-acetylgalactosaminidases were identified by screening bacterial libraries in 2007, allowing improved conversion of B and the first useful conversions of A-type RBCs, although under constrained conditions. In 2019, screening of a metagenomic library derived from the feces of an AB donor enabled discovery of a significantly more efficient two-enzyme system, involving a GalNAc deacetylase and a galactosaminidase, for A conversion. This promising system works well both in standard conditions and in whole blood. We discuss remaining challenges and opportunities for the use of such enzymes in blood conversion and organ transplantation.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/metabolismo , Biotecnología , Donantes de Sangre , Glicósido Hidrolasas/metabolismo , Animales , Bacterias/enzimología , Biocatálisis , Biotecnología/métodos , Transfusión Sanguínea , Hexosaminidasas/metabolismo , Humanos , Modelos Moleculares , alfa-Galactosidasa/metabolismoRESUMEN
Plant specialized metabolism serves as a rich resource of biologically active molecules for drug discovery. The acylated flavonol glycoside montbretin A (MbA) and its precursor myricetin 3-O-(6'-O-caffeoyl)-glucosyl rhamnoside (mini-MbA) are potent inhibitors of human pancreatic α-amylase and are being developed as drug candidates to treat type-2 diabetes. MbA occurs in corms of the ornamental plant montbretia (Crocosmia x crocosmiiflora), but a system for large-scale MbA production is currently unavailable. Biosynthesis of MbA from the flavonol myricetin and MbA accumulation occur during early stages of corm development. We established myricetin 3-O-rhamnoside (MR), myricetin 3-O-glucosyl rhamnoside (MRG), and mini-MbA as the first three intermediates of MbA biosynthesis. Contrasting the transcriptomes of young and old corms revealed differentially expressed UDP-sugar-dependent glycosyltransferases (UGTs) and BAHD-acyltransferases (BAHD-ATs). UGT77B2 and UGT709G2 catalyze the consecutive glycosylation of myricetin to produce MR and of MR to give MRG, respectively. In addition, two BAHD-ATs, CcAT1 and CcAT2, catalyze the acylation of MRG to complete the formation of mini-MbA. Transcript profiles of UGT77B2, UGT709G2, CcAT1, and CcAT2 during corm development matched the metabolite profile of MbA accumulation. Expression of these enzymes in wild tobacco (Nicotiana benthamiana) resulted in the formation of a surrogate mini-MbA, validating the potential for metabolic engineering of mini-MbA in a heterologous plant system.
Asunto(s)
Aciltransferasas/metabolismo , Flavonas/metabolismo , Glicosiltransferasas/metabolismo , Nicotiana/metabolismo , Trisacáridos/metabolismo , Aciltransferasas/genética , Glicosiltransferasas/genética , Proteínas de Plantas/metabolismoRESUMEN
ß-N-Acetylhexosaminidases (HexNAcases) are versatile biocatalysts that cleave terminal N-acetylhexosamine units from various glycoconjugates. Established strategies to generate glycoside-forming versions of the wild type enzymes rely on the mutation of their catalytic residues; however, successful examples of synthetically useful HexNAcase mutants are scarce. In order to expand the range of HexNAcases available as targets for enzyme engineering, we functionally screened a metagenomic library derived from a human gut microbiome. From a pool of hits, we characterized four of the more active candidates by sequence analysis and phylogenetic mapping, and found that they all belonged to CAZy family GH20. After detailed kinetic analysis and characterization of their substrate specificities, active site mutants were generated which resulted in the identification of two new thioglycoligases. BvHex E294A and AsHex E301A catalyzed glycosyl transfer to all three of the 3-, 4- and 6-thio-N-acetylglucosaminides (thio-GlcNAcs) that were tested. Both mutant enzymes also catalyzed glycosyl transfer to a cysteine-containing variant of the model peptide Tab1, with AsHex E301A also transferring GlcNAc onto a thiol-containing protein. This work illustrates how large scale functional screening of expressed gene libraries allows the relatively rapid development of useful new glycoside-forming mutants of HexNAcases, expanding the pool of biocatalysts for carbohydrate synthesis.
Asunto(s)
AcetilglucosaminidasaRESUMEN
As part of a search for a substrate for droplet-based microfluidic screening assay of α-N-acetylgalactosaminidases, spectral and physical characteristics of a series of coumarin derivatives were measured. From among these a new coumarin-based fluorophore, Jericho Blue, was selected as having optimal characteristics for our screen. A reliable method for the challenging synthesis of coumarin glycosides of α-GalNAc was then developed and demonstrated with nine examples. The α-GalNAc glycoside of Jericho Blue prepared in this way was shown to function well under screening conditions.
Asunto(s)
Cumarinas/química , Cumarinas/síntesis química , Pruebas de Enzimas/instrumentación , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Dispositivos Laboratorio en un Chip , alfa-N-Acetilgalactosaminidasa/metabolismo , Técnicas de Química Sintética , Especificidad por Sustrato , alfa-N-Acetilgalactosaminidasa/químicaRESUMEN
Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4-20 µM-1·s-1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or ß-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Galactósidos/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Simportadores/metabolismo , Sitios de Unión , Transporte Biológico Activo , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Colorantes Fluorescentes , Galactosa/química , Galactosa/metabolismo , Galactósidos/química , Cinética , Ligandos , Modelos Moleculares , Estructura Molecular , Proteínas de Transporte de Monosacáridos/química , Periplasma/metabolismo , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Simportadores/químicaRESUMEN
Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of N-acetylglucosamine (GlcNAc, A) and d-glucosamine (GlcN, D). Production of well-defined CHOS-mixtures, or even pure CHOS, with specific lengths and sugar compositions, is of great interest since these oligosaccharides have interesting bioactivities. While direct chemical synthesis of CHOS is not straightforward, chemo-enzymatic approaches have shown some promise. We have used engineered glycoside hydrolases to catalyze oligomerization of activated DA building blocks through transglycosylation reactions. The building blocks were generated from readily available (GlcNAc)2-para-nitrophenol through deacetylation of the nonreducing end sugar with a recombinantly expressed deacetylase from Aspergillus niger (AnCDA9). This approach, using a previously described hyper-transglycosylating variant of ChiA from Serratia marcescens (SmChiA) and a newly generated transglycosylating variant of Chitinase D from Serratia proteamaculans (SpChiD), led to production of CHOS containing up to ten alternating D and A units [(DA)2, (DA)3, (DA)4, and (DA)5]. The most abundant compounds were purified and characterized. Finally, we demonstrate that (DA)3 generated in this study may serve as a specific inhibitor of the human chitotriosidase. Inhibition of this enzyme has been suggested as a therapeutic strategy against systemic sclerosis.
Asunto(s)
Quitina/análogos & derivados , Oligosacáridos/biosíntesis , Oligosacáridos/síntesis química , Acetilglucosamina/química , Aspergillus niger/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Carbohidratos , Quitina/biosíntesis , Quitina/síntesis química , Quitinasas/genética , Quitinasas/metabolismo , Cristalografía por Rayos X , Glucosamina/química , Hexosaminidasas/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligosacáridos/química , Serratia/enzimología , Serratia/genética , Serratia marcescens/enzimología , Serratia marcescens/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
α-Linked GalNAc (α-GalNAc) is most notably found at the nonreducing terminus of the blood type-determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans. However, despite their ubiquity in saccharolytic microbe-rich environments such as the human gut, relatively few α-N-acetylgalactosaminidases are known. Here, to discover and characterize novel microbial enzymes that hydrolyze α-GalNAc, we screened small-insert libraries containing metagenomic DNA from the human gut microbiome. Using a simple fluorogenic glycoside substrate, we identified and characterized a glycoside hydrolase 109 (GH109) that is active on blood type A-antigen, along with a new subfamily of glycoside hydrolase 31 (GH31) that specifically cleaves the initial α-GalNAc from mucin-type O-glycans. This represents a new activity in this GH family and a potentially useful new enzyme class for analysis or modification of O-glycans on protein or cell surfaces.