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1.
Biochimie ; 163: 50-57, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078584

RESUMO

Acylpeptidyl-oligopeptidase (AOP), which has been recently identified as a novel enzyme in a periodontopathic bacterium, Porphyromonas gingivalis, removes di- and tri-peptides from N-terminally acylated polypeptides, with a preference for hydrophobic P1-position amino acid residues. To validate enzymatic properties of AOP, characteristics of two bacterial orthologues from Bacteroides dorei (BdAOP), a Gram-negative intestinal rod, and Lysinibacillus sphaericus (LsAOP), a Gram-positive soil rod, were determined. Like P. gingivalis AOP (PgAOP), two orthologues more efficiently hydrolyzed N-terminal acylated peptidyl substrates than non-acylated ones. Optimal pH was shifted from 7.0 to 8.9 for N-acylated to 8.5-9.5 for non-acylated substrates, indicating preference for non-charged hydrophobic N-terminal residues. Hydrophobic P1- and P2-position preferences were common in the three AOPs, although PgAOP preferred Leu and the others preferred Phe at the P1 position. In vitro mutagenesis demonstrated that Phe647 in PgAOP was responsible for the P1 Leu preference. In addition, bacterial AOPs commonly liberated acetyl-Ser1-Tyr2 from α-melanocyte-stimulating hormone. Taken together, although these three bacterial AOPs exhibited some variations in biochemical properties, the present study demonstrated the existence of a group of exopeptidases that preferentially liberates mainly dipeptides from N-terminally acylated polypeptides with a preference for hydrophobic P1 and P2-position residues.


Assuntos
Peptídeo Hidrolases/metabolismo , Porphyromonas gingivalis/enzimologia , Bacillaceae/enzimologia , Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Hidrólise , Cinética , Peptídeo Hidrolases/efeitos dos fármacos , Especificidade por Substrato
2.
Biomol NMR Assign ; 13(1): 213-218, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30734154

RESUMO

Bacteroides ovatus is a member of the human gut microbiota. The importance of this microbial consortium involves the degradation of complex dietary glycans mainly conferred by glycoside hydrolases. In this study we focus on one such catabolic glycoside hydrolase from B. ovatus. The enzyme, termed BoMan26A, is a ß-mannanase that takes part in the hydrolytic degradation of galactomannans. The crystal structure of BoMan26A has previously been determined to reveal a TIM-barrel like fold, but the relation between the protein structure and the mode of substrate processing has not yet been studied. Here we report residue-specific assignments for 95% of the 344 backbone amides of BoMan26A. The assignments form the basis for future studies of the relationship between substrate interactions and protein dynamics. In particular, the potential role of loops adjacent to glycan binding sites is of interest for such studies.


Assuntos
Bacteroides/enzimologia , Microbioma Gastrointestinal , Ressonância Magnética Nuclear Biomolecular , beta-Manosidase/química , Isótopos de Carbono , Humanos , Isótopos de Nitrogênio , Estrutura Secundária de Proteína , Prótons
3.
Int J Mol Sci ; 19(12)2018 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-30544819

RESUMO

The form and physiology of Bradyrhizobium diazoefficiens after the decline of symbiotic nitrogen fixation has been characterized. Proteomic analyses showed that post-symbiotic B. diazoefficiens underwent metabolic remodeling as well-defined groups of proteins declined, increased or remained unchanged from 56 to 119 days after planting, suggesting a transition to a hemibiotrophic-like lifestyle. Enzymatic analysis showed distinct patterns in both the cytoplasm and the periplasm. Similar to the bacteroid, the post-symbiotic bacteria rely on a non-citric acid cycle supply of succinate and, although viable, they did not demonstrate the ability to grow within the senescent nodule.


Assuntos
Bacteroides/metabolismo , Bradyrhizobium/metabolismo , Proteômica/métodos , Nódulos Radiculares de Plantas/microbiologia , Soja/crescimento & desenvolvimento , Soja/microbiologia , Simbiose , Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , Bacteroides/isolamento & purificação , Hidroxibutiratos/metabolismo , Leghemoglobina/metabolismo , Periplasma/metabolismo , Poliésteres/metabolismo
4.
Appl Microbiol Biotechnol ; 102(12): 5133-5147, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29680900

RESUMO

Sulfide detoxification can be catalyzed by ancient membrane-bound flavoproteins, sulfide:quinone oxidoreductases (Sqr), which have important roles in sulfide homeostasis and sulfide-dependent energy conservation processes by transferring electrons from sulfide to respiratory or photosynthetic membrane electron flow. Sqr enzymes have been categorized into six groups. Several members of the groups I, II, III, and V are well-known, but type IV and VI Sqrs are, as yet, uncharacterized or hardly characterized at all. Here, we report detailed characterization of a type VI sulfide:quinone oxidoreductase (TrSqrF) from a purple sulfur bacterium, Thiocapsa roseopersicina. Phylogenetic analysis classified this enzyme in a special group composed of SqrFs of endosymbionts, while a weaker relationship could be observed with SqrF of Chlorobaculum tepidum which is the only type VI enzyme characterized so far. Directed mutagenesis experiments showed that TrSqrF contributed substantially to the sulfide:quinone oxidoreductase activity of the membranes. Expression of the sqrF gene could be induced by sulfide. Homologous recombinant TrSqrF protein was expressed and purified from the membranes of a SqrF-deleted T. roseopersicina strain. The purified protein contains redox-active covalently bound FAD cofactor. The recombinant TrSqrF enzyme catalyzes sulfur-dependent quinone reduction and prefers ubiquinone-type quinone compounds. Kinetic parameters of TrSqrF show that the affinity of the enzyme is similar to duroquinone and decylubiquinone, but the reaction has substantially lower activation energy with decylubiquinone, indicating that the quinone structure has an effect on the catalytic process. TrSqrF enzyme affinity for sulfide is low, therefore, in agreement with the gene expressional analyis, SqrF could play a role in energy-conserving sulfide oxidation at high sulfide concentrations. TrSqrF is a good model enzyme for the subgroup of type VI Sqrs of endosymbionts and its characterization might provide deeper insight into the molecular details of the ancient, anoxic, energy-gaining processes using sulfide as an electron donor.


Assuntos
Bacteroides/enzimologia , Quinona Redutases/metabolismo , Bacteroides/classificação , Regulação Bacteriana da Expressão Gênica , Oxirredução , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfetos/metabolismo
5.
Enzyme Microb Technol ; 114: 29-32, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29685350

RESUMO

Two GH43 ß-xylosidases, RS223-BX from a rice straw metagenomic library, and BoXA from Bacteroides ovatus, that share similar amino acid sequences (81% identical) and 19 of 20 active-site residues, were compared by using site-directed mutagenesis of Asp and His residues implicated in metal binding. Thus, RS223-BX is strongly activated by divalent-metal cations and the previously published X-ray structure of this enzyme shows that a Ca2+ cation is chelated by an active-site Asp carboxyl group and an active-site His. Mutation to Ala causes 90% loss of activity for the Asp mutant and 98% loss of activity for the His mutant, indicating their importance to catalysis. For the other enzyme (BoXA), mutation to Ala causes 20% loss of activity for the His mutant and 40% gain of activity for the Asp mutant, indicating the lack of importance for activity of the native residues and the lack of metal-dependency, given that the Asp residue occupies the active site to secure the metal cation in known metal ion dependent GH43 xylosidases. The high activity of the BoXA mutants compared to that of the analogous RS223-BX mutants further undermines the possibility that BoXA maintains a tightly bound metal cofactor resistant to EDTA extraction. The results strengthen our conclusion that the very similar proteins differ in one being metal ion dependent and one not.


Assuntos
Proteínas de Bactérias/química , Bacteroides/enzimologia , Cálcio/metabolismo , Oryza/enzimologia , Proteínas de Plantas/química , Xilosidases/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides/química , Bacteroides/genética , Biocatálise , Cálcio/química , Domínio Catalítico , Ativação Enzimática , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oryza/química , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Xilosidases/genética , Xilosidases/metabolismo
6.
Infect Dis (Lond) ; 50(5): 372-380, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29303023

RESUMO

BACKGROUND: The species of the Bacteroides fragilis group are important components of human microbiota, but as opportunistic pathogens they can be the causative agents of severe infections. METHODS: The major aims of our investigation were the evaluation of the susceptibility of 400 different Hungarian B. fragilis group isolates to 10 antibiotics by the agar dilution method, the comparison of our resistance data with previous national and international antibiotic resistance data and the comparison of present data in regional aspect. The MIC-values on 10 antibiotics of all the strains were determined with the agar dilution method by CLSI. The presence of the cfiA gene in Division II B. fragilis strains was confirmed by RT-PCR. RESULTS: We detected a relatively high resistance rate of ampicillin, moxifloxacin, clindamycin and tetracycline, but amoxicillin/clavulanic acid, metronidazole, tigecycline and chloramphenicol showed excellent activity. In this study, we found that 6.75% of the isolates were resistant to cefoxitin and 7% to meropenem, while 8.58% of our B. fragilis strains harboured the cfiA gene. Most of the meropenem resistant strains were isolated in one of the participating centres. In the case of meropenem, cefoxitin, clindamycin and high-level-ampicillin-resistant strains, we found significant regional differences. DISCUSSION: Most of the results of our study were concordant with previous national and international data, with the exception of amoxicillin/clavulanic acid, cefoxitin and meropenem. CONCLUSIONS: Our study highlighted the importance of the periodic monitoring of the antimicrobial susceptibility of Bacteroides species providing important information for the appropriate therapy.


Assuntos
Antibacterianos/farmacologia , Infecções por Bacteroides/epidemiologia , Infecções por Bacteroides/microbiologia , Bacteroides/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Inquéritos e Questionários , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Combinação Amoxicilina e Clavulanato de Potássio/farmacologia , Ampicilina/farmacologia , Bacteroides/enzimologia , Bacteroides/genética , Bacteroides/isolamento & purificação , Infecções por Bacteroides/tratamento farmacológico , Criança , Pré-Escolar , Feminino , Humanos , Hungria/epidemiologia , Imipenem/farmacologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Adulto Jovem , beta-Lactamases/biossíntese , beta-Lactamases/efeitos dos fármacos
7.
Biochimie ; 147: 25-35, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29080830

RESUMO

Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which liberate dipeptides from the N-termini of polypeptides along with the penultimate hydrophobic and acidic residues, respectively. Their specificities are primarily defined by a single amino acid residue, Gly673 in DPP7 and Arg673 in DPP11 (numbering for Porphyromonas gingivalis DPP11). Bacterial species in the phyla Proteobacteria and Bacteroidetes generally possess one gene for each, while Bacteroides species exceptionally possess three genes, one gene as DPP7 and two genes as DPP11, annotated based on the full-length similarities. In the present study, we aimed to characterize the above-mentioned Bacteroides S46 DPPs. A recombinant protein of the putative DPP11 gene BF9343_2924 from Bacteroides fragilis harboring Gly673 exhibited DPP7 activity by hydrolyzing Leu-Leu-4-methylcoumaryl-7-amide (MCA). Another gene, BF9343_2925, as well as the Bacteroides vulgatus gene (BVU_2252) with Arg673 was confirmed to encode DPP11. These results demonstrated that classification of S46 peptidase is enforceable by the S1 essential residues. Bacteroides DPP11 showed a decreased level of activity towards the substrates, especially with P1-position Glu. Findings of 3D structural modeling indicated three potential amino acid substitutions responsible for the reduction, one of which, Asn650Thr substitution, actually recovered the hydrolyzing activity of Leu-Glu-MCA. On the other hand, the gene currently annotated as DPP7 carrying Gly673 from B. fragilis (BF9343_0130) and Bacteroides ovatus (Bovatus_03382) did not hydrolyze any of the examined substrates. The existence of a phylogenic branch of these putative Bacteroides DPP7 genes classified by the C-terminal conserved region (Ser571-Leu700) strongly suggests that Bacteroides species expresses a DPP with an unknown property. In conclusion, the genus Bacteroides exceptionally expresses three S46-family members; authentic DPP7, a new subtype of DPP11 with substantially reduced specificity for Glu, and a third group of S46 family members.


Assuntos
Bacteroides/enzimologia , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Sequência de Aminoácidos , Hidrólise , Especificidade da Espécie
8.
J Microbiol ; 55(11): 892-899, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29076071

RESUMO

Clostridium difficile infection (CDI) is one of the most common nosocomial infections. Dysbiosis of the gut microbiota due to consumption of antibiotics is a major contributor to CDI. Recently, fecal microbiota transplantation (FMT) has been applied to treat CDI. However, FMT has important limitations including uncontrolled exposure to pathogens and standardization issues. Therefore, it is necessary to evaluate alternative treatment methods, such as bacteriotherapy, as well as the mechanism through which beneficial bacteria inhibit the growth of C. difficile. Here, we report bile acid-mediated inhibition of C. difficile by Bacteroides strains which can produce bile salt hydrolase (BSH). Bacteroides strains are not commonly used to treat CDI; however, as they comprise a large proportion of the intestinal microbiota, they can contribute to bile acid-mediated inhibition of C. difficile. The inhibitory effect on C. difficile growth increased with increasing bile acid concentration in the presence of Bacteroides ovatus SNUG 40239. Furthermore, this inhibitory effect on C. difficile growth was significantly attenuated when bile acid availability was reduced by cholestyramine, a bile acid sequestrant. The findings of this study are important due to the discovery of a new bacterial strain that in the presence of available bile acids inhibits growth of C. difficile. These results will facilitate development of novel bacteriotherapy strategies to control CDI.


Assuntos
Bacteroides/enzimologia , Ácidos e Sais Biliares/metabolismo , Clostridium difficile/efeitos dos fármacos , Clostridium difficile/crescimento & desenvolvimento , Hidrolases/metabolismo , Antibiose , Bacteroides/metabolismo , Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/farmacologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/terapia , Fezes/microbiologia , Microbioma Gastrointestinal , Humanos
9.
Cell ; 171(3): 655-667.e17, 2017 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-29053971

RESUMO

The gut microbiota contributes to the development of normal immunity but, when dysregulated, can promote autoimmunity through various non-antigen-specific effects on pathogenic and regulatory lymphocytes. Here, we show that an integrase expressed by several species of the gut microbial genus Bacteroides encodes a low-avidity mimotope of the pancreatic ß cell autoantigen islet-specific glucose-6-phosphatase-catalytic-subunit-related protein (IGRP206-214). Studies in germ-free mice monocolonized with integrase-competent, integrase-deficient, and integrase-transgenic Bacteroides demonstrate that the microbial epitope promotes the recruitment of diabetogenic CD8+ T cells to the gut. There, these effectors suppress colitis by targeting microbial antigen-loaded, antigen-presenting cells in an integrin ß7-, perforin-, and major histocompatibility complex class I-dependent manner. Like their murine counterparts, human peripheral blood T cells also recognize Bacteroides integrase. These data suggest that gut microbial antigen-specific cytotoxic T cells may have therapeutic value in inflammatory bowel disease and unearth molecular mimicry as a novel mechanism by which the gut microbiota can regulate normal immune homeostasis. PAPERCLIP.


Assuntos
Autoantígenos/imunologia , Bacteroides/imunologia , Colite/imunologia , Microbioma Gastrointestinal , Glucose-6-Fosfatase/imunologia , Adulto , Animais , Bacteroides/classificação , Bacteroides/enzimologia , Colite/microbiologia , Feminino , Glucose-6-Fosfatase/genética , Humanos , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Mimetismo Molecular , Linfócitos T/imunologia
10.
J Mol Biol ; 429(16): 2509-2527, 2017 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-28669823

RESUMO

Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s-1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98Å resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases.


Assuntos
Bacteroides/enzimologia , Esterases/química , Esterases/metabolismo , Xilosidases/metabolismo , Sequência de Aminoácidos , Ácidos Cafeicos/metabolismo , Cromatografia em Gel , Ácidos Cumáricos/metabolismo , Cristalografia por Raios X , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , Multimerização Proteica , Alinhamento de Sequência , Especificidade por Substrato , Xilanos/metabolismo
11.
Proc Natl Acad Sci U S A ; 114(27): 7037-7042, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28630303

RESUMO

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.


Assuntos
Bacteroides/enzimologia , Microbioma Gastrointestinal , Glicosaminoglicanos/química , Bacteroides/genética , Calorimetria , Carboidratos/química , Catálise , Cristalografia por Raios X , Citoplasma/enzimologia , Carboidratos da Dieta , Heparina/química , Heparitina Sulfato/química , Humanos , Microscopia de Fluorescência , Mutação , Oligossacarídeos/química , Polissacarídeo-Liase/química , Polissacarídeos/química , Sulfatases/química , Enxofre/química
12.
J Bacteriol ; 199(10)2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28242723

RESUMO

CTnDOT is an integrated conjugative element found in Bacteroides species. CTnDOT contains and transfers antibiotic resistance genes. The element integrates into and excises from the host chromosome via a Holliday junction (HJ) intermediate as part of a site-specific recombination mechanism. The CTnDOT integrase, IntDOT, is a tyrosine recombinase with core-binding, catalytic, and amino-terminal (N) domains. Unlike well-studied tyrosine recombinases, such as lambda integrase (Int), IntDOT is able to resolve Holliday junctions containing heterology (mismatched bases) between the sites of strand exchange. All known natural isolates of CTnDOT contain mismatches in the overlap region between the sites of strand exchange. Previous work showed that IntDOT was unable to resolve synthetic Holliday junctions containing mismatched bases to products in the absence of the arm-type sites and a DNA-bending protein. We constructed synthetic HJs with the arm-type sites and tested them with the Bacteroides host factor (BHFa). We found that the addition of BHFa stimulated resolution of HJ intermediates with mismatched overlap regions to products. In addition, the L1 site is required for directionality of the reaction, particularly when the HJ contains mismatches. BHFa is required for product formation when the overlap region contains mismatches, and it stimulates resolution to products when the overlap region is identical. Without this DNA bending, the N domain of IntDOT is likely unable to bind the L1 arm-type site. These findings suggest that BHFa bends DNA into the necessary conformation for the higher-order complexes, including the L1 site, that are required for product formation.IMPORTANCE CTnDOT is a mobile element that carries antibiotic resistance genes and moves by site-selective recombination and subsequent conjugation. The recombination reaction is catalyzed by an integrase IntDOT that is a member of the tyrosine recombinase family. The reaction proceeds through ordered strand exchanges that generate a Holliday junction (HJ) intermediate. Unlike other tyrosine recombinases, IntDOT can resolve HJs containing mismatched bases in the overlap region in vivo, as is the case under natural conditions. However, HJ intermediates including only IntDOT core-type sites cannot be resolved to products if the HJ intermediate contains mismatched bases. We added arm-type sites in cis and in trans to the HJ intermediates and the protein BHFa to study the requirements for higher-order nucleoprotein complexes.


Assuntos
Bacteroides/enzimologia , Bacteroides/metabolismo , Pareamento Incorreto de Bases , DNA Cruciforme/metabolismo , Recombinases/metabolismo , Bacteroides/genética , DNA/genética , DNA/metabolismo , Fatores Hospedeiros de Integração/metabolismo
13.
J Agric Food Chem ; 65(12): 2530-2539, 2017 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-28252294

RESUMO

Lactulose (4-O-ß-d-galactopyranosyl-d-fructofuranose) is a prebiotic sugar derived from the milk sugar lactose (4-O-ß-d-galactopyranosyl-d-glucopyranose). In our study we observed for the first time that known cellobiose 2-epimerases (CEs; EC 5.1.3.11) from mesophilic microorganisms were generally able to catalyze the isomerization reaction of lactose into lactulose. Commonly, CEs catalyze the C2-epimerization of d-glucose and d-mannose moieties at the reducing end of ß-1,4-glycosidic-linked oligosaccharides. Thus, epilactose (4-O-ß-d-galactopyranosyl-d-mannopyranose) is formed with lactose as substrate. So far, only four CEs, exclusively from thermophilic microorganisms, have been reported to additionally catalyze the isomerization reaction of lactose into lactulose. The specific isomerization activity of the seven CEs in this study ranged between 8.7 ± 0.1 and 1300 ± 37 pkat/mg. The results indicate that very likely all CEs are able to catalyze both the epimerization as well as the isomerization reaction, whereby the latter is performed at a comparatively much lower reaction rate.


Assuntos
Proteínas de Bactérias/química , Carboidratos Epimerases/química , Celobiose/metabolismo , Lactulose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , Bacteroides/genética , Biocatálise , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Celobiose/química , Estabilidade Enzimática , Flavobacterium/enzimologia , Flavobacterium/genética , Lactose/metabolismo , Lactulose/química
14.
Chemistry ; 23(13): 3197-3205, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28092124

RESUMO

Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases.


Assuntos
Bacteroides/enzimologia , Domínio Catalítico , Cellvibrio/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Bacteroides/química , Bacteroides/metabolismo , Cellvibrio/química , Cellvibrio/metabolismo , Hidrólise , Oligossacarídeos/síntese química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Técnicas de Síntese em Fase Sólida , Especificidade por Substrato , Xilanos/síntese química , Xilanos/química , Xilanos/metabolismo , Xilosidases/química , Xilosidases/metabolismo
15.
J Struct Biol ; 197(3): 227-235, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27890857

RESUMO

Bacteria from the human gut are equipped with an arsenal of carbohydrate-active enzymes that degrade dietary and host-derived glycans. In this study, we present the 2.5Å resolution crystal structure of a member (GH39wh2) from the human gut bacteria Bacteroides cellulosilyticus WH2 representative of a new subgroup within family GH39. Together with 6 other GHs, GH39wh2 belongs to a polysaccharide utilization locus (PUL) that could be involved in detecting, binding and hydrolysing a specific carbohydrate species from the intestinal tract. GH39wh2 shares a similar architecture as other members of family GH39 dominated by a typical (ß/α)8-barrel fold harboring the catalytic residues and decorated by ß-sandwich accessory domains. The GH39wh2 structure unveils an atypical shallow groove rather than a deep pocket due to drastic rearrangements in surface loops surrounding the catalytic interface. These structural adaptations seem to favour recognition of large branched substrates and may explain the lack of activity of GH39wh2 toward small xylose-based and other typical substrates from GH39 members, emphasizing the molecular diversity within the GH39 family. A phylogenetic analysis of the entire GH39 family assigns GH39wh2 as a new subgroup, consistent with the extensive remodelling of the active site region that may confer new substrate specificity toward a complex glycan chain.


Assuntos
Bacteroides/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Domínio Catalítico , Biologia Computacional , Cristalografia por Raios X , Microbioma Gastrointestinal , Glicosídeo Hidrolases/classificação , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato
16.
J Biol Chem ; 292(1): 229-243, 2017 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-27872187

RESUMO

A recently identified polysaccharide utilization locus (PUL) from Bacteroides ovatus ATCC 8483 is transcriptionally up-regulated during growth on galacto- and glucomannans. It encodes two glycoside hydrolase family 26 (GH26) ß-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan-binding proteins, confirmed by ß-mannan affinity electrophoresis. When this PUL was deleted, B. ovatus was no longer able to grow on locust bean galactomannan. BoMan26A primarily formed mannobiose from mannan polysaccharides. BoMan26B had higher activity on galactomannan with a high degree of galactosyl substitution and was shown to be endo-acting generating a more diverse mixture of oligosaccharides, including mannobiose. Of the two ß-mannanases, only BoMan26B hydrolyzed galactoglucomannan. A crystal structure of BoMan26A revealed a similar structure to the exo-mannobiohydrolase CjMan26C from Cellvibrio japonicus, with a conserved glycone region (-1 and -2 subsites), including a conserved loop closing the active site beyond subsite -2. Analysis of cellular location by immunolabeling and fluorescence microscopy suggests that BoMan26B is surface-exposed and associated with the outer membrane, although BoMan26A and BoGal36A are likely periplasmic. In light of the cellular location and the biochemical properties of the two characterized ß-mannanases, we propose a scheme of sequential action by the glycoside hydrolases encoded by the ß-mannan PUL and involved in the ß-mannan utilization pathway in B. ovatus. The outer membrane-associated BoMan26B initially acts on the polysaccharide galactomannan, producing comparably large oligosaccharide fragments. Galactomanno-oligosaccharides are further processed in the periplasm, degalactosylated by BoGal36A, and subsequently hydrolyzed into mainly mannobiose by the ß-mannanase BoMan26A.


Assuntos
Bacteroides/enzimologia , Mananas/metabolismo , Polissacarídeos/metabolismo , beta-Manosidase/química , beta-Manosidase/metabolismo , Catálise , Cristalografia por Raios X , Hidrólise , Conformação Proteica , Especificidade por Substrato
17.
Appl Biochem Biotechnol ; 182(1): 250-260, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27854035

RESUMO

Divalent metal-activated glycoside hydrolase family 43 (GH43) ß-xylosidases have been found to have high k cat/K m for xylooligosaccharides and may demonstrate high efficacy in industrial reactors digesting hemicellulose. By searching an amino acid database, we found a Bacteroides ovatus GH43 ß-xylosidase termed BoXA that is 81% identical in overall amino acid sequence to a GH43, divalent metal-activated ß-xylosidase with high k cat/K m, and also it has 19 of 20 residues in the active site conserved. However, unlike its metal-activated homolog, the B. ovatus enzyme does not lose activity after extensive EDTA treatment nor does it gain activity by addition of divalent metal ions. Thus, either it cannot be activated by divalent metal or it maintains a tightly bound, non-exchangeable metal ion. At 25 °C and pH 6.0, the k cat is 69 s-1 for xylobiose and k cat/K m is 210 s-1 mM-1 for xylotriose, with the latter being 0.7 that of the highest known value. The determined K i for D-glucose is 4.9 M, which is the highest known for a ß-xylosidase. The enzyme has potential utility operating in bioreactors digesting plant biomass.


Assuntos
Proteínas de Bactérias/química , Bacteroides/química , Glucuronatos/química , Oligossacarídeos/química , Xilosidases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Bacteroides/enzimologia , Bacteroides/genética , Sequência de Bases , Domínio Catalítico , Cátions Bivalentes , Dissacarídeos/química , Ácido Edético/química , Ativação Enzimática , Expressão Gênica , Glucose/química , Concentração de Íons de Hidrogênio , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Temperatura Ambiente , Trissacarídeos/química , Xilosidases/genética , Xilosidases/isolamento & purificação
18.
Cell Host Microbe ; 20(6): 709-715, 2016 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-27916477

RESUMO

Renal disease is growing in prevalence and has striking co-morbidities with metabolic and cardiovascular disease. Indoxyl sulfate (IS) is a toxin that accumulates in plasma when kidney function declines and contributes to the progression of chronic kidney disease. IS derives exclusively from the gut microbiota. Bacterial tryptophanases convert tryptophan to indole, which is absorbed and modified by the host to produce IS. Here, we identify a widely distributed family of tryptophanases in the gut commensal Bacteroides and find that deleting this gene eliminates the production of indole in vitro. By altering the status or abundance of the Bacteroides tryptophanase, we can modulate IS levels in gnotobiotic mice and in the background of a conventional murine gut community. Our results demonstrate that it is possible to control host IS levels by targeting the microbiota and suggest a possible strategy for treating renal disease.


Assuntos
Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/microbiologia , Indicã/metabolismo , Indicã/toxicidade , Ração Animal , Animais , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Bacteroides/enzimologia , Bacteroides/genética , Dieta , Modelos Animais de Doenças , Progressão da Doença , Microbioma Gastrointestinal/genética , Engenharia Genética , Vida Livre de Germes/efeitos dos fármacos , Humanos , Indóis/metabolismo , Metagenoma , Camundongos , Microbiota/genética , Insuficiência Renal Crônica , Toxinas Biológicas/biossíntese , Toxinas Biológicas/urina , Triptofano/metabolismo , Triptofanase/metabolismo
19.
Chem Commun (Camb) ; 52(74): 11096-9, 2016 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-27546776

RESUMO

Bacteroides vulgatus is a member of the human microbiota whose abundance is increased in patients with Crohn's disease. We show that a B. vulgatus glycoside hydrolase from the carbohydrate active enzyme family GH123, BvGH123, is an N-acetyl-ß-galactosaminidase that acts with retention of stereochemistry, and, through a 3-D structure in complex with Gal-thiazoline, provide evidence in support of a neighbouring group participation mechanism.


Assuntos
Bacteroides/enzimologia , beta-N-Acetil-Galactosaminidase/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Sítios de Ligação , Domínio Catalítico , Doença de Crohn/etiologia , Doença de Crohn/microbiologia , Humanos , Simulação de Dinâmica Molecular , Estereoisomerismo , Especificidade por Substrato , Tiazóis/química , Tiazóis/metabolismo , beta-N-Acetil-Galactosaminidase/química
20.
Open Biol ; 6(7)2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27466444

RESUMO

The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta)genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a ß-glucosidase, and two α-l-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , Glucanos/metabolismo , Xilanos/metabolismo , Arabinose/análogos & derivados , Arabinose/química , Bacteroides/química , Cristalografia por Raios X , Trato Gastrointestinal/microbiologia , Humanos , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Xilosidases/química , beta-Glucosidase/química
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