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1.
Cell ; 185(14): 2495-2509.e11, 2022 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-35764090

RESUMEN

Plant fibers in byproduct streams produced by non-harsh food processing methods represent biorepositories of diverse, naturally occurring, and physiologically active biomolecules. To demonstrate one approach for their characterization, mass spectrometry of intestinal contents from gnotobiotic mice, plus in vitro studies, revealed liberation of N-methylserotonin from orange fibers by human gut microbiota members including Bacteroides ovatus. Functional genomic analyses of B. ovatus strains grown under permissive and non-permissive N-methylserotonin "mining" conditions revealed polysaccharide utilization loci that target pectins whose expression correlate with strain-specific liberation of this compound. N-methylserotonin, orally administered to germ-free mice, reduced adiposity, altered liver glycogenesis, shortened gut transit time, and changed expression of genes that regulate circadian rhythm in the liver and colon. In human studies, dose-dependent, orange-fiber-specific fecal accumulation of N-methylserotonin positively correlated with levels of microbiome genes encoding enzymes that digest pectic glycans. Identifying this type of microbial mining activity has potential therapeutic implications.


Asunto(s)
Citrus sinensis , Microbioma Gastrointestinal , Animales , Citrus sinensis/metabolismo , Fibras de la Dieta , Microbioma Gastrointestinal/fisiología , Vida Libre de Gérmenes , Humanos , Ratones , Pectinas/metabolismo , Polisacáridos/metabolismo , Serotonina/análogos & derivados
2.
Cell ; 179(1): 59-73.e13, 2019 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-31539500

RESUMEN

Development of microbiota-directed foods (MDFs) that selectively increase the abundance of beneficial human gut microbes, and their expressed functions, requires knowledge of both the bioactive components of MDFs and the mechanisms underlying microbe-microbe interactions. Here, gnotobiotic mice were colonized with a defined consortium of human-gut-derived bacterial strains and fed different combinations of 34 food-grade fibers added to a representative low-fiber diet consumed in the United States. Bioactive carbohydrates in fiber preparations targeting particular Bacteroides species were identified using community-wide quantitative proteomic analyses of bacterial gene expression coupled with forward genetic screens. Deliberate manipulation of community membership combined with administration of retrievable artificial food particles, consisting of paramagnetic microscopic beads coated with dietary polysaccharides, disclosed the contributions of targeted species to fiber degradation. Our approach, including the use of bead-based biosensors, defines nutrient-harvesting strategies that underlie, as well as alleviate, competition between Bacteroides and control the selectivity of MDF components.


Asunto(s)
Bacteroides/genética , Fibras de la Dieta/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Vida Libre de Gérmenes/fisiología , Interacciones Microbianas/efectos de los fármacos , Polisacáridos/farmacología , Proteómica/métodos , Animales , Dieta/métodos , Fibras de la Dieta/metabolismo , Heces/microbiología , Microbioma Gastrointestinal/fisiología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Polisacáridos/metabolismo
4.
Cell ; 167(5): 1339-1353.e21, 2016 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-27863247

RESUMEN

Despite the accepted health benefits of consuming dietary fiber, little is known about the mechanisms by which fiber deprivation impacts the gut microbiota and alters disease risk. Using a gnotobiotic mouse model, in which animals were colonized with a synthetic human gut microbiota composed of fully sequenced commensal bacteria, we elucidated the functional interactions between dietary fiber, the gut microbiota, and the colonic mucus barrier, which serves as a primary defense against enteric pathogens. We show that during chronic or intermittent dietary fiber deficiency, the gut microbiota resorts to host-secreted mucus glycoproteins as a nutrient source, leading to erosion of the colonic mucus barrier. Dietary fiber deprivation, together with a fiber-deprived, mucus-eroding microbiota, promotes greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. Our work reveals intricate pathways linking diet, the gut microbiome, and intestinal barrier dysfunction, which could be exploited to improve health using dietary therapeutics.


Asunto(s)
Fibras de la Dieta/administración & dosificación , Microbioma Gastrointestinal , Mucosa Intestinal/microbiología , Animales , Citrobacter rodentium/fisiología , Colitis/microbiología , Colon/microbiología , Susceptibilidad a Enfermedades , Infecciones por Enterobacteriaceae/microbiología , Escherichia coli , Femenino , Vida Libre de Gérmenes , Humanos , Masculino , Ratones , Mucina 2/genética
5.
Nature ; 625(7993): 157-165, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38093016

RESUMEN

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Here we analyse biospecimens from a randomized, controlled trial of a microbiome-directed complementary food (MDCF-2) that produced superior rates of weight gain compared with a calorically more dense conventional ready-to-use supplementary food in 12-18-month-old Bangladeshi children with moderate acute malnutrition4. We reconstructed 1,000 bacterial genomes (metagenome-assembled genomes (MAGs)) from the faecal microbiomes of trial participants, identified 75 MAGs of which the abundances were positively associated with ponderal growth (change in weight-for-length Z score (WLZ)), characterized changes in MAG gene expression as a function of treatment type and WLZ response, and quantified carbohydrate structures in MDCF-2 and faeces. The results reveal that two Prevotella copri MAGs that are positively associated with WLZ are the principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilizing the component glycans of MDCF-2. The predicted specificities of carbohydrate-active enzymes expressed by their polysaccharide-utilization loci are correlated with (1) the in vitro growth of Bangladeshi P. copri strains, possessing varying degrees of polysaccharide-utilization loci and genomic conservation with these MAGs, in defined medium containing different purified glycans representative of those in MDCF-2, and (2) the levels of faecal carbohydrate structures in the trial participants. These associations suggest that identifying bioactive glycan structures in MDCFs metabolized by growth-associated bacterial taxa will help to guide recommendations about their use in children with acute malnutrition and enable the development of additional formulations.


Asunto(s)
Alimentos , Microbioma Gastrointestinal , Desnutrición , Polisacáridos , Humanos , Lactante , Bacterias/genética , Bangladesh , Peso Corporal/genética , Heces/microbiología , Microbioma Gastrointestinal/fisiología , Genoma Bacteriano/genética , Desnutrición/microbiología , Metagenoma/genética , Polisacáridos/metabolismo , Aumento de Peso
6.
Proc Natl Acad Sci U S A ; 120(39): e2311422120, 2023 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-37733741

RESUMEN

Understanding how members of the human gut microbiota prioritize nutrient resources is one component of a larger effort to decipher the mechanisms defining microbial community robustness and resiliency in health and disease. This knowledge is foundational for development of microbiota-directed therapeutics. To model how bacteria prioritize glycans in the gut, germfree mice were colonized with 13 human gut bacterial strains, including seven saccharolytic Bacteroidaceae species. Animals were fed a Western diet supplemented with pea fiber. After community assembly, an inducible CRISPR-based system was used to selectively and temporarily reduce the absolute abundance of Bacteroides thetaiotaomicron or B. cellulosilyticus by 10- to 60-fold. Each knockdown resulted in specific, reproducible increases in the abundances of other Bacteroidaceae and dynamic alterations in their expression of genes involved in glycan utilization. Emergence of these "alternate consumers" was associated with preservation of community saccharolytic activity. Using an inducible system for CRISPR base editing in vitro, we disrupted translation of transporters critical for utilizing dietary polysaccharides in Phocaeicola vulgatus, a B. cellulosilyticus knockdown-responsive taxon. In vitro and in vivo tests of the resulting P. vulgatus mutants allowed us to further characterize mechanisms associated with its increased fitness after knockdown. In principle, the approach described can be applied to study utilization of a range of nutrients and to preclinical efforts designed to develop therapeutic strategies for precision manipulation of microbial communities.


Asunto(s)
Bacteroides thetaiotaomicron , Bacteroides , Humanos , Animales , Ratones , Bacteroides/genética , Polisacáridos , Bacteroides thetaiotaomicron/genética , Bioensayo , Dieta Occidental
7.
Semin Cell Dev Biol ; 134: 112-124, 2023 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-35307283

RESUMEN

In brown algae, the extracellular matrix (ECM) and its constitutive polymers play crucial roles in specialized functions, including algal growth and development. In this review we offer an integrative view of ECM construction in brown algae. We briefly report the chemical composition of its main constituents, and how these are interlinked in a structural model. We examine the ECM assembly at the tissue and cell level, with consideration on its structure in vivo and on the putative subcellular sites for the synthesis of its main constituents. We further discuss the biosynthetic pathways of two major polysaccharides, alginates and sulfated fucans, and the progress made beyond the candidate genes with the biochemical validation of encoded proteins. Key enzymes involved in the elongation of the glycan chains are still unknown and predictions have been made at the gene level. Here, we offer a re-examination of some glycosyltransferases and sulfotransferases from published genomes. Overall, our analysis suggests novel investigations to be performed at both the cellular and biochemical levels. First, to depict the location of polysaccharide structures in tissues. Secondly, to identify putative actors in the ECM synthesis to be functionally studied in the future.


Asunto(s)
Phaeophyceae , Phaeophyceae/genética , Phaeophyceae/química , Phaeophyceae/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Genoma , Matriz Extracelular/metabolismo
8.
J Biol Chem ; 299(4): 103038, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36806678

RESUMEN

The Carbohydrate-Active Enzyme classification groups enzymes that breakdown, assemble, or decorate glycans into protein families based on sequence similarity. The glycoside hydrolases (GH) are arranged into over 170 enzyme families, with some being very large and exhibiting distinct activities/specificities towards diverse substrates. Family GH31 is a large family that contains more than 20,000 sequences with a wide taxonomic diversity. Less than 1% of GH31 members are biochemically characterized and exhibit many different activities that include glycosidases, lyases, and transglycosidases. This diversity of activities limits our ability to predict the activities and roles of GH31 family members in their host organism and our ability to exploit these enzymes for practical purposes. Here, we established a subfamily classification using sequence similarity networks that was further validated by a structural analysis. While sequence similarity networks provide a sequence-based separation, we obtained good segregation between activities among the subfamilies. Our subclassification consists of 20 subfamilies with sixteen subfamilies containing at least one characterized member and eleven subfamilies that are monofunctional based on the available data. We also report the biochemical characterization of a member of the large subfamily 2 (GH31_2) that lacked any characterized members: RaGH31 from Rhodoferax aquaticus is an α-glucosidase with activity on a range of disaccharides including sucrose, trehalose, maltose, and nigerose. Our subclassification provides improved predictive power for the vast majority of uncharacterized proteins in family GH31 and highlights the remaining sequence space that remains to be functionally explored.


Asunto(s)
Glicósido Hidrolasas , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Filogenia , Polisacáridos/metabolismo , Proteínas , Especificidad por Sustrato , Betaproteobacteria/enzimología , Familia de Multigenes
9.
Environ Microbiol ; 26(5): e16624, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38757353

RESUMEN

Laminarin, a ß(1,3)-glucan, serves as a storage polysaccharide in marine microalgae such as diatoms. Its abundance, water solubility and simple structure make it an appealing substrate for marine bacteria. Consequently, many marine bacteria have evolved strategies to scavenge and decompose laminarin, employing carbohydrate-binding modules (CBMs) as crucial components. In this study, we characterized two previously unassigned domains as laminarin-binding CBMs in multimodular proteins from the marine bacterium Christiangramia forsetii KT0803T, thereby introducing the new laminarin-binding CBM families CBM102 and CBM103. We identified four CBM102s in a surface glycan-binding protein (SGBP) and a single CBM103 linked to a glycoside hydrolase module from family 16 (GH16_3). Our analysis revealed that both modular proteins have an elongated shape, with GH16_3 exhibiting greater flexibility than SGBP. This flexibility may aid in the recognition and/or degradation of laminarin, while the constraints in SGBP could facilitate the docking of laminarin onto the bacterial surface. Exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea showed that both laminarin-binding CBM families are widespread among marine Bacteroidota. The high protein abundance of CBM102- and CBM103-containing proteins during phytoplankton blooms further emphasizes their significance in marine laminarin utilization.


Asunto(s)
Proteínas Bacterianas , Glucanos , Fitoplancton , Glucanos/metabolismo , Fitoplancton/metabolismo , Fitoplancton/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Bacteroidetes/metabolismo , Bacteroidetes/genética , Eutrofización , Diatomeas/metabolismo , Diatomeas/genética , Receptores de Superficie Celular
10.
PLoS Comput Biol ; 19(8): e1010881, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37585436

RESUMEN

The deluge of genomic data raises various challenges for computational protein annotation. The definition of superfamilies, based on conserved folds, or of families, showing more recent homology signatures, allow a first categorization of the sequence space. However, for precise functional annotation or the identification of the unexplored parts within a family, a division into subfamilies is essential. As curators of an expert database, the Carbohydrate Active Enzymes database (CAZy), we began, more than 15 years ago, to manually define subfamilies based on phylogeny reconstruction. However, facing the increasing amount of sequence and functional data, we required more scalable and reproducible methods. The recently popularized sequence similarity networks (SSNs), allows to cope with very large families and computation of many subfamily schemes. Still, the choice of the optimal SSN subfamily scheme only relies on expert knowledge so far, without any data-driven guidance from within the network. In this study, we therefore decided to investigate several network properties to determine a criterion which can be used by curators to evaluate the quality of subfamily assignments. The performance of the closeness centrality criterion, a network property to indicate the connectedness within the network, shows high similarity to the decisions of expert curators from eight distinct protein families. Closeness centrality also suggests that in some cases multiple levels of subfamilies could be possible, depending on the granularity of the research question, while it indicates when no subfamily emerged in some family evolution. We finally used closeness centrality to create subfamilies in four families of the CAZy database, providing a finer functional annotation and highlighting subfamilies without biochemically characterized members for potential future discoveries.


Asunto(s)
Glicósido Hidrolasas , Proteínas , Proteínas/genética , Proteínas/química , Secuencia de Aminoácidos , Glicósido Hidrolasas/química , Anotación de Secuencia Molecular , Filogenia , Bases de Datos de Proteínas
11.
Appl Microbiol Biotechnol ; 108(1): 415, 2024 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-38990377

RESUMEN

Currently, the main α-amylase family GH13 has been divided into 47 subfamilies in CAZy, with new subfamilies regularly emerging. The present in silico study was performed to highlight the groups, represented by the maltogenic amylase from Thermotoga neapolitana and the α-amylase from Haloarcula japonica, which are worth of creating their own new GH13 subfamilies. This enlarges functional annotation and thus allows more precise prediction of the function of putative proteins. Interestingly, those two share certain sequence features, e.g. the highly conserved cysteine in the second conserved sequence region (CSR-II) directly preceding the catalytic nucleophile, or the well-preserved GQ character of the end of CSR-VII. On the other hand, the two groups bear also specific and highly conserved positions that distinguish them not only from each other but also from representatives of remaining GH13 subfamilies established so far. For the T. neapolitana maltogenic amylase group, it is the stretch of residues at the end of CSR-V highly conserved as L-[DN]. The H. japonica α-amylase group can be characterized by a highly conserved [WY]-[GA] sequence at the end of CSR-II. Other specific sequence features include an almost fully conserved aspartic acid located directly preceding the general acid/base in CSR-III or well-preserved glutamic acid in CSR-IV. The assumption that these two groups represent two mutually related, but simultaneously independent GH13 subfamilies has been supported by phylogenetic analysis as well as by comparison of tertiary structures. The main α-amylase family GH13 has thus been expanded by two novel subfamilies GH13_48 and GH13_49. KEY POINTS: • In silico analysis of two groups of family GH13 members with characterized representatives • Identification of certain common, but also some specific sequence features in seven CSRs • Creation of two novel subfamilies-GH13_48 and GH13_49 within the CAZy database.


Asunto(s)
Filogenia , alfa-Amilasas , alfa-Amilasas/genética , alfa-Amilasas/metabolismo , alfa-Amilasas/química , Secuencia de Aminoácidos , Secuencia Conservada , Alineación de Secuencia
12.
Cell Mol Life Sci ; 80(8): 232, 2023 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-37500984

RESUMEN

Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular α-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular α-amylase, BoGH13ASus, is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13ASus is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13ASus CBM98-CBM48-GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13ASus structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an α-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13ASus specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut.


Asunto(s)
Bacteroides , alfa-Amilasas , Humanos , Bacteroides/metabolismo , Almidón/metabolismo , Polisacáridos/metabolismo
13.
Nucleic Acids Res ; 50(D1): D571-D577, 2022 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-34850161

RESUMEN

Thirty years have elapsed since the emergence of the classification of carbohydrate-active enzymes in sequence-based families that became the CAZy database over 20 years ago, freely available for browsing and download at www.cazy.org. In the era of large scale sequencing and high-throughput Biology, it is important to examine the position of this specialist database that is deeply rooted in human curation. The three primary tasks of the CAZy curators are (i) to maintain and update the family classification of this class of enzymes, (ii) to classify sequences newly released by GenBank and the Protein Data Bank and (iii) to capture and present functional information for each family. The CAZy website is updated once a month. Here we briefly summarize the increase in novel families and the annotations conducted during the last 8 years. We present several important changes that facilitate taxonomic navigation, and allow to download the entirety of the annotations. Most importantly we highlight the considerable amount of work that accompanies the analysis and report of biochemical data from the literature.


Asunto(s)
Carbohidratos/química , Bases de Datos de Ácidos Nucleicos , Bases de Datos de Proteínas , Enzimas/química , Carbohidratos/clasificación , Activación Enzimática/genética , Enzimas/clasificación , Humanos
14.
Nature ; 544(7648): 65-70, 2017 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-28329766

RESUMEN

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.


Asunto(s)
Bacteroides thetaiotaomicron/enzimología , Bacteroides thetaiotaomicron/metabolismo , Biocatálisis , Tracto Gastrointestinal/microbiología , Glicósido Hidrolasas/metabolismo , Pectinas/química , Pectinas/metabolismo , Bacteroides thetaiotaomicron/crecimiento & desarrollo , Boratos/química , Boratos/metabolismo , Dominio Catalítico , Microbioma Gastrointestinal , Glicósido Hidrolasas/química , Glicósido Hidrolasas/clasificación , Humanos , Modelos Moleculares , Especificidad por Sustrato
16.
Proc Natl Acad Sci U S A ; 116(13): 6063-6068, 2019 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-30850540

RESUMEN

Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein sequences remains one of the main challenges in modern biology. As in all classes of proteins, the growing number of predicted carbohydrate-active enzymes (CAZymes) has not been accompanied by a systematic and accurate attribution of function. Taking advantage of the CAZy database, which groups CAZymes into families and subfamilies based on amino acid similarities, we recombinantly produced 564 proteins selected from subfamilies without any biochemically characterized representatives, from distant relatives of characterized enzymes and from nonclassified proteins that show little similarity with known CAZymes. Screening these proteins for activity on a wide collection of carbohydrate substrates led to the discovery of 13 CAZyme families (two of which were also discovered by others during the course of our work), revealed three previously unknown substrate specificities, and assigned a function to 25 subfamilies.


Asunto(s)
Metabolismo de los Hidratos de Carbono , Enzimas/genética , Análisis de Secuencia de Proteína , Secuencia de Aminoácidos , Animales , Metabolismo de los Hidratos de Carbono/genética , Enzimas/metabolismo , Genómica/métodos , Humanos , Polisacáridos/metabolismo , Análisis de Secuencia de ADN , Relación Estructura-Actividad
17.
J Biol Chem ; 294(44): 15973-15986, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31501245

RESUMEN

Glycoside hydrolase family (GH) 16 comprises a large and taxonomically diverse family of glycosidases and transglycosidases that adopt a common ß-jelly-roll fold and are active on a range of terrestrial and marine polysaccharides. Presently, broadly insightful sequence-function correlations in GH16 are hindered by a lack of a systematic subfamily structure. To fill this gap, we have used a highly scalable protein sequence similarity network analysis to delineate nearly 23,000 GH16 sequences into 23 robust subfamilies, which are strongly supported by hidden Markov model and maximum likelihood molecular phylogenetic analyses. Subsequent evaluation of over 40 experimental three-dimensional structures has highlighted key tertiary structural differences, predominantly manifested in active-site loops, that dictate substrate specificity across the GH16 evolutionary landscape. As for other large GH families (i.e. GH5, GH13, and GH43), this new subfamily classification provides a roadmap for functional glycogenomics that will guide future bioinformatics and experimental structure-function analyses. The GH16 subfamily classification is publicly available in the CAZy database. The sequence similarity network workflow used here, SSNpipe, is freely available from GitHub.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Fúngicas/química , Glicósido Hidrolasas/genética , Filogenia , Análisis de Secuencia de Proteína/métodos , Algoritmos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Dominio Catalítico , Evolución Molecular , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/genética , Glicómica/métodos , Glicósido Hidrolasas/química , Glicósido Hidrolasas/clasificación
18.
Nucleic Acids Res ; 46(D1): D677-D683, 2018 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-29088389

RESUMEN

The Polysaccharide Utilization Loci (PUL) database was launched in 2015 to present PUL predictions in ∼70 Bacteroidetes species isolated from the human gastrointestinal tract, as well as PULs derived from the experimental data reported in the literature. In 2018 PULDB offers access to 820 genomes, sampled from various environments and covering a much wider taxonomical range. A Krona dynamic chart was set up to facilitate browsing through taxonomy. Literature surveys now allows the presentation of the most recent (i) PUL repertoires deduced from RNAseq large-scale experiments, (ii) PULs that have been subjected to in-depth biochemical analysis and (iii) new Carbohydrate-Active enzyme (CAZyme) families that contributed to the refinement of PUL predictions. To improve PUL visualization and genome browsing, the previous annotation of genes encoding CAZymes, regulators, integrases and SusCD has now been expanded to include functionally relevant protein families whose genes are significantly found in the vicinity of PULs: sulfatases, proteases, ROK repressors, epimerases and ATP-Binding Cassette and Major Facilitator Superfamily transporters. To cope with cases where susCD may be absent due to incomplete assemblies/split PULs, we present 'CAZyme cluster' predictions. Finally, a PUL alignment tool, operating on the tagged families instead of amino-acid sequences, was integrated to retrieve PULs similar to a query of interest. The updated PULDB website is accessible at www.cazy.org/PULDB_new/.


Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteroidetes/metabolismo , Bases de Datos de Compuestos Químicos , Bases de Datos Genéticas , Genes Bacterianos , Operón/genética , Polisacáridos/metabolismo , Proteínas Bacterianas/genética , Bacteroidetes/clasificación , Bacteroidetes/genética , Transporte Biológico/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Chlorobi/clasificación , Chlorobi/genética , Chlorobi/metabolismo , Metabolismo Energético/genética , Enzimas/genética , Enzimas/metabolismo , Evolución Molecular , Fibrobacteres/clasificación , Fibrobacteres/genética , Fibrobacteres/metabolismo , Regulación Bacteriana de la Expresión Génica , Anotación de Secuencia Molecular , Familia de Multigenes , ARN Bacteriano/genética , Alineación de Secuencia , Especificidad de la Especie
19.
Proc Natl Acad Sci U S A ; 114(19): 4936-4941, 2017 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-28396425

RESUMEN

The human gut microbiota use complex carbohydrates as major nutrients. The requirement for an efficient glycan degrading systems exerts a major selection pressure on this microbial community. Thus, we propose that these bacteria represent a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis, we focused on enzymes that hydrolyze rhamnosidic bonds, as cleavage of these linkages is chemically challenging and there is a paucity of information on l-rhamnosidases. Here we screened the activity of enzymes derived from the human gut microbiota bacterium Bacteroides thetaiotaomicron, which are up-regulated in response to rhamnose-containing glycans. We identified an α-l-rhamnosidase, BT3686, which is the founding member of a glycoside hydrolase (GH) family, GH145. In contrast to other rhamnosidases, BT3686 cleaved l-Rha-α1,4-d-GlcA linkages through a retaining double-displacement mechanism. The crystal structure of BT3686 showed that the enzyme displayed a type A seven-bladed ß-propeller fold. Mutagenesis and crystallographic studies, including the structure of BT3686 in complex with the reaction product GlcA, revealed a location for the active site among ß-propeller enzymes cited on the posterior surface of the rhamnosidase. In contrast to the vast majority of GH, the catalytic apparatus of BT3686 does not comprise a pair of carboxylic acid residues but, uniquely, a single histidine functions as the only discernable catalytic amino acid. Intriguingly, the histidine, His48, is not invariant in GH145; however, when engineered into structural homologs lacking the imidazole residue, α-l-rhamnosidase activity was established. The potential contribution of His48 to the catalytic activity of BT3686 is discussed.


Asunto(s)
Proteínas Bacterianas/química , Bacteroides thetaiotaomicron/enzimología , Glicósido Hidrolasas/química , Proteínas Bacterianas/genética , Bacteroides thetaiotaomicron/genética , Cristalografía por Rayos X , Glicósido Hidrolasas/genética , Humanos , Mutagénesis
20.
Proc Natl Acad Sci U S A ; 114(27): 7037-7042, 2017 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-28630303

RESUMEN

The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron, a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS, and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides, indicating that the model developed is of generic relevance to this important microbial community.


Asunto(s)
Bacteroides/enzimología , Microbioma Gastrointestinal , Glicosaminoglicanos/química , Bacteroides/genética , Calorimetría , Carbohidratos/química , Catálisis , Cristalografía por Rayos X , Citoplasma/enzimología , Carbohidratos de la Dieta , Heparina/química , Heparitina Sulfato/química , Humanos , Microscopía Fluorescente , Mutación , Oligosacáridos/química , Polisacárido Liasas/química , Polisacáridos/química , Sulfatasas/química , Azufre/química
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