RESUMEN

Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/ß hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome.

Asunto(s)

Proteínas Bacterianas , Filogenia , Piromyces , Rumen , Rumen/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Piromyces/enzimología , Piromyces/genética , Esterasas/genética , Esterasas/química , Esterasas/metabolismo , Esterasas/clasificación , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fibrobacter/enzimología , Fibrobacter/genética , Fibrobacter/clasificación , Dominio Catalítico , Ruminococcus/enzimología , Ruminococcus/genética , Ruminococcus/clasificación , Modelos Moleculares

RESUMEN

D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+, and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization. The half-life of the best variant E268R-EKL16 at 50 °C was dramatically increased 30-fold to 135.3 h, and it maintained 90 % of its activity after 13 consecutive reaction cycles. Additionally, we identified that metal ions played a key role in stabilizing the tetramer structure of Rum55, and the dependence on metal ions for E268R-EKL16 was significantly reduced. This study provides a useful route for improving the thermostability of DAEs, opening up new possibilities for the industrial production of D-allulose.

Asunto(s)

Estabilidad de Enzimas , Ingeniería de Proteínas , Ruminococcus , Ruminococcus/enzimología , Ruminococcus/genética , Ingeniería de Proteínas/métodos , Péptidos/química , Péptidos/metabolismo , Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Cinética , Modelos Moleculares , Fructosa/metabolismo , Fructosa/química

RESUMEN

Humans, like all mammals, depend on the gut microbiome for digestion of cellulose, the main component of plant fiber. However, evidence for cellulose fermentation in the human gut is scarce. We have identified ruminococcal species in the gut microbiota of human populations that assemble functional multienzymatic cellulosome structures capable of degrading plant cell wall polysaccharides. One of these species, which is strongly associated with humans, likely originated in the ruminant gut and was subsequently transferred to the human gut, potentially during domestication where it underwent diversification and diet-related adaptation through the acquisition of genes from other gut microbes. Collectively, these species are abundant and widespread among ancient humans, hunter-gatherers, and rural populations but are rare in populations from industrialized societies thus indicating potential disappearance in response to the westernized lifestyle.

Asunto(s)

Celulosa , Fibras de la Dieta , Microbioma Gastrointestinal , Ruminococcus , Humanos , Celulosa/metabolismo , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiología , Ruminococcus/clasificación , Ruminococcus/enzimología , Ruminococcus/genética , Fibras de la Dieta/metabolismo , Filogenia , Desarrollo Industrial

RESUMEN

Cellulosomes are highly complex cell-bound multi-enzymatic nanomachines used by anaerobes to break down plant carbohydrates. The genome sequence of Ruminococcus flavefaciens revealed a remarkably diverse cellulosome composed of more than 200 cellulosomal enzymes. Here we provide a detailed biochemical characterization of a highly elaborate R. flavefaciens cellulosomal enzyme containing an N-terminal dockerin module, which anchors the enzyme into the multi-enzyme complex through binding of cohesins located in non-catalytic cell-bound scaffoldins, and three tandemly repeated family 16 glycoside hydrolase (GH16) catalytic domains. The DNA sequence encoding the three homologous catalytic domains was cloned and hyper-expressed in Escherichia coli BL21 (DE3) cells. SDS-PAGE analysis of purified His6 tag containing RfGH16_21 showed a single soluble protein of molecular size ~89 kDa, which was in agreement with the theoretical size, 89.3 kDa. The enzyme RfGH16_21 exhibited activity over a wide pH range (pH 5.0-8.0) and a broad temperature range (50-70 °C), displaying maximum activity at an optimum pH of 7.0 and optimum temperature of 55 °C. Substrate specificity analysis of RfGH16_21 revealed maximum activity against barley ß-d-glucan (257 U mg-1) followed by lichenan (247 U mg-1), but did not show significant activity towards other tested polysaccharides, suggesting that it is specifically a ß-1,3-1,4-endoglucanase. TLC analysis revealed that RfGH16_21 hydrolyses barley ß-d-glucan to cellotriose, cellotetraose and a higher degree of polymerization of gluco-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a fairly high, active and thermostable bacterial endo-glucanase which may find considerable biotechnological potentials.

Asunto(s)

Proteínas Bacterianas/química , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Ruminococcus/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Estabilidad de Enzimas , Glucanos/metabolismo , Glicósido Hidrolasas/genética , Concentración de Iones de Hidrógeno , Familia de Multigenes , Dominios Proteicos , Ruminococcus/química , Ruminococcus/genética , Especificidad por Sustrato , Temperatura

RESUMEN

BACKGROUND: Yaks are able to utilize the gastrointestinal microbiota to digest plant materials. Although the cellulolytic bacteria in the yak rumen have been reported, there is still limited information on the diversity of the major microorganisms and putative carbohydrate-metabolizing enzymes for the degradation of complex lignocellulosic biomass in its gut ecosystem. RESULTS: Here, this study aimed to decode biomass-degrading genes and genomes in the yak fecal microbiota using deep metagenome sequencing. A comprehensive catalog comprising 4.5 million microbial genes from the yak feces were established based on metagenomic assemblies from 92 Gb sequencing data. We identified a full spectrum of genes encoding carbohydrate-active enzymes, three-quarters of which were assigned to highly diversified enzyme families involved in the breakdown of complex dietary carbohydrates, including 120 families of glycoside hydrolases, 25 families of polysaccharide lyases, and 15 families of carbohydrate esterases. Inference of taxonomic assignments to the carbohydrate-degrading genes revealed the major microbial contributors were Bacteroidaceae, Ruminococcaceae, Rikenellaceae, Clostridiaceae, and Prevotellaceae. Furthermore, 68 prokaryotic genomes were reconstructed and the genes encoding glycoside hydrolases involved in plant-derived polysaccharide degradation were identified in these uncultured genomes, many of which were novel species with lignocellulolytic capability. CONCLUSIONS: Our findings shed light on a great diversity of carbohydrate-degrading enzymes in the yak gut microbial community and uncultured species, which provides a useful genetic resource for future studies on the discovery of novel enzymes for industrial applications.

Asunto(s)

Esterasas/genética , Microbioma Gastrointestinal/genética , Glicósido Hidrolasas/genética , Metagenómica , Consorcios Microbianos/genética , Polisacárido Liasas/genética , Rumen/microbiología , Animales , Bacteroidaceae/enzimología , Bacteroidaceae/genética , Bacteroidaceae/aislamiento & purificación , Bacteroidetes/enzimología , Bacteroidetes/genética , Bacteroidetes/aislamiento & purificación , Metabolismo de los Hidratos de Carbono , Bovinos , Clostridiaceae/enzimología , Clostridiaceae/genética , Clostridiaceae/aislamiento & purificación , Esterasas/clasificación , Esterasas/aislamiento & purificación , Esterasas/metabolismo , Heces/microbiología , Expresión Génica , Variación Genética , Glicósido Hidrolasas/clasificación , Glicósido Hidrolasas/aislamiento & purificación , Glicósido Hidrolasas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Lignina/metabolismo , Metagenoma , Metagenómica/métodos , Polisacárido Liasas/clasificación , Polisacárido Liasas/aislamiento & purificación , Polisacárido Liasas/metabolismo , Prevotella/enzimología , Prevotella/genética , Prevotella/aislamiento & purificación , Rumen/enzimología , Ruminococcus/enzimología , Ruminococcus/genética , Ruminococcus/aislamiento & purificación

RESUMEN

D-Allose is a rare sugar, can be used as an ingredient in a range of foods and dietary supplements, has alimentary activities, especially excellent anti-cancer effects and used in assisting cancer chemotherapy and radiotherapy, etc. To develop a simple and low-cost process for D-allose production, a one-pot enzymatic process using the substrate of D-fructose, and the recombinant enzymes of D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) was developed. These enzymes were cloned from Ruminococcus sp. and B. subtilis, respectively, successfully expressed in E. coli, extracted and immobilized using anion exchange resin and amino resin, respectively. The mass ratio of D-fructose, D-psicose and D-allose was 6.6:2.4:1.0 when the reaction reached equilibrium after 5 h of reaction. Using the low-cost substrate of D-fructose, the reusable immobilized enzymes and the one-pot reaction, the production process is simplified and the production cost is decreased. In addition, to simplify the enzyme extraction and immobilization processes, new methods for enzyme capture and immobilization were developed especially for DPE immobilization. This is the first report for one-pot D-allose production using immobilized L-RhI and DPE.

Asunto(s)

Isomerasas Aldosa-Cetosa/química , Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Carbohidrato Epimerasas/química , Fructosa/química , Glucosa/síntesis química , Ruminococcus/enzimología , Isomerasas Aldosa-Cetosa/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Carbohidrato Epimerasas/genética , Glucosa/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ruminococcus/genética

RESUMEN

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with ß-(1,4)-linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble ß-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.

Asunto(s)

Biomasa , Glicósido Hidrolasas/metabolismo , Ascomicetos/enzimología , Sitios de Unión , Dominio Catalítico , Bases de Datos de Proteínas , Glucanos/química , Glucanos/metabolismo , Hidrólisis , Cinética , Mananos/metabolismo , Simulación de Dinámica Molecular , Ruminococcus/enzimología , Especificidad por Sustrato , Xilanos/química , Xilanos/metabolismo

RESUMEN

Sialic acid (N-acetylneuraminic acid (Neu5Ac)) is commonly found in the terminal location of colonic mucin glycans where it is a much-coveted nutrient for gut bacteria, including Ruminococcus gnavus. R. gnavus is part of the healthy gut microbiota in humans, but it is disproportionately represented in diseases. There is therefore a need to understand the molecular mechanisms that underpin the adaptation of R. gnavus to the gut. Previous in vitro research has demonstrated that the mucin-glycan-foraging strategy of R. gnavus is strain dependent and is associated with the expression of an intramolecular trans-sialidase, which releases 2,7-anhydro-Neu5Ac, rather than Neu5Ac, from mucins. Here, we unravelled the metabolism pathway of 2,7-anhydro-Neu5Ac in R. gnavus that is underpinned by the exquisite specificity of the sialic transporter for 2,7-anhydro-Neu5Ac and by the action of an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac, which then becomes a substrate of a Neu5Ac-specific aldolase. Having generated an R. gnavus nan-cluster deletion mutant that lost the ability to grow on sialylated substrates, we showed that-in gnotobiotic mice colonized with R. gnavus wild-type (WT) and mutant strains-the fitness of the nan mutant was significantly impaired, with a reduced ability to colonize the mucus layer. Overall, we revealed a unique sialic acid pathway in bacteria that has important implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease.

Asunto(s)

Adaptación Fisiológica , Microbioma Gastrointestinal/fisiología , Moco/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Ruminococcus/metabolismo , Animales , Clostridiales , Glicoproteínas , Humanos , Redes y Vías Metabólicas/genética , Redes y Vías Metabólicas/fisiología , Ratones , Ratones Endogámicos C57BL , Mucinas/metabolismo , Ácido N-Acetilneuramínico/análogos & derivados , Neuraminidasa , Oxo-Ácido-Liasas/metabolismo , Polisacáridos/metabolismo , Proteínas Recombinantes , Ruminococcus/enzimología , Ruminococcus/genética

RESUMEN

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.

Asunto(s)

Celulasa/metabolismo , Ruminococcus/enzimología , beta-Manosidasa/metabolismo , Secuencia de Aminoácidos/genética , Celulasa/biosíntesis , Celulasa/química , Celulasa/genética , Celulosomas/enzimología , Quelantes , Cromatografía en Capa Delgada , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Galactanos/química , Galactanos/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Mananos/química , Mananos/metabolismo , Oligosacáridos/química , Gomas de Plantas/química , Gomas de Plantas/metabolismo , Ruminococcus/genética , Especificidad por Sustrato , Temperatura , beta-Manosidasa/química , beta-Manosidasa/genética

RESUMEN

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ribonucleasas/metabolismo , Ruminococcus/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Conformación de Ácido Nucleico , Dominios Proteicos , Procesamiento Postranscripcional del ARN , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Guía de Kinetoplastida/genética , Ribonucleasas/química , Ribonucleasas/genética , Ruminococcus/enzimología

RESUMEN

Ruminococcus is one of the keystone bacteria of the human colonic microbiota and is highly specific for utilization of resistant starch via formation of amylosomes. Here, we present the characteristics of an extracellular amylase, Rbamy5, in Ruminococcus bromii. In an in silico study, it showed low homology with any other known amylases, but it was evolutionarily classified as a GH13_36 subfamily intermediary amylase. Recombinant Rbamy5 exhibited maximum activity toward amylose (384 ±â€¯26 U·mg-1) over soluble starch (254 ±â€¯3 U·mg-1), amylopectin (46.1 ±â€¯2.6 U·mg-1) and pullulan (72.5 ±â€¯0.41 U·mg-1) at 45 °C and pH 5.0. It was also able to degrade small substrates such as maltotriose (G3), maltotetraose (G4), and maltopentaose (G5) into maltose (G2). Despite lacking a specific N-terminal domain, Rbamy5 opened the cyclodextrin ring, which resembles those of neopullulanase. Moreover, it accumulated short α-(1 → 6)-branched maltooligosaccharides from soluble starch and maltosyl-ß-cyclodextrin (G2-ß-CD), while panose was solely derived from pullulan. These results suggest that Rbamy5 may have a role to provide branched maltooligosaccharides to stimulate the growth of beneficial microorganisms in the human intestine.

Asunto(s)

Glicósido Hidrolasas/química , Ruminococcus/enzimología , Ruminococcus/genética , alfa-Amilasas/química , alfa-Amilasas/genética , Secuencia de Aminoácidos , Clonación Molecular , Activación Enzimática , Espacio Extracelular/enzimología , Glicósido Hidrolasas/metabolismo , Hidrólisis , Filogenia , Análisis de Secuencia de ADN , Análisis Espectral , Especificidad por Sustrato , alfa-Amilasas/aislamiento & purificación , alfa-Amilasas/metabolismo

RESUMEN

From the rice-based culture of Malbranchea flavorosea, three new compounds namely flavoroseoside B (5-desoxy-5-chloro-flavoroseoside) (2), 4-hydroxy-2-O-α-ribofuranosyl-5-methylacetophenone (3), and (S)-3,4-dihydro-3-(1H-indol-3-ylmethyl)-4-methyl-1H-1,4-benzodiazepine-2,5-dione (4), along with three known compounds, rosigenin (5), massarilactone B (6), and riboxylarinol B (7) were obtained. The structures were determined by spectroscopic methods. Compound 4 and its synthetic analog 3,4-dihydro-3-(1H-indol-3-ylmethyl)-1-methyl-1H-1,4-benzodiazepine-2,5-dione (9) inhibited the activity of Ruminococus obeum α-glucosidase enzyme. Molecular docking and dynamic studies revealed that compounds 4 and 9 might bind to this α-glucosidase at the catalytic center. Phylogenetic analysis using internal transcribed spacer region revealed that Malbranchea flavorosea ATCC 34529 is related to Myxotrichum spp.

Asunto(s)

Ascomicetos/metabolismo , Inhibidores de Glicósido Hidrolasas/metabolismo , Inhibidores de Glicósido Hidrolasas/farmacología , Inhibidores de Glicósido Hidrolasas/química , Simulación del Acoplamiento Molecular , Estructura Molecular , Ruminococcus/enzimología , Saccharomyces cerevisiae/enzimología , alfa-Glucosidasas/metabolismo

RESUMEN

ADP-glucose pyrophosphorylase from Firmicutes is encoded by two genes (glgC and glgD) leading to a heterotetrameric protein structure, unlike those in other bacterial phyla. The enzymes from two groups of Firmicutes, Bacillales and Lactobacillales, present dissimilar kinetic and regulatory properties. Nevertheless, no ADP-glucose pyrophosphorylase from Clostridiales, the third group in Firmicutes, has been characterized. For this reason, we cloned the glgC and glgD genes from Ruminococcus albus Different quaternary forms of the enzyme (GlgC, GlgD, and GlgC/GlgD) were purified to homogeneity and their kinetic parameters were analyzed. We observed that GlgD is an inactive monomer when expressed alone but increased the catalytic efficiency of the heterotetramer (GlgC/GlgD) compared to the homotetramer (GlgC). The heterotetramer is regulated by fructose-1,6-bisphosphate, phosphoenolpyruvate, and NAD(P)H. The first characterization of the Bacillales enzyme suggested that heterotetrameric ADP-glucose pyrophosphorylases from Firmicutes were unregulated. Our results, together with data from Lactobacillales, indicate that heterotetrameric Firmicutes enzymes are mostly regulated. Thus, the ADP-glucose pyrophosphorylase from Bacillales seems to have distinctive insensitivity to regulation.IMPORTANCE The enzymes involved in glycogen synthesis from Firmicutes have been less characterized in comparison with other bacterial groups. We performed kinetic and regulatory characterization of the ADP-glucose pyrophosphorylase from Ruminococcus albus Our results showed that this protein that belongs to different groups from Firmicutes (Bacillales, Lactobacillales, and Clostridiales) presents dissimilar features. This study contributes to the understanding of how this critical enzyme for glycogen biosynthesis is regulated in the Firmicutes group, whereby we propose that these heterotetrameric enzymes, with the exception of Bacillales, are allosterically regulated. Our results provide a better understanding of the evolutionary relationship of this enzyme family in Firmicutes.

Asunto(s)

Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Ruminococcus/enzimología , Ruminococcus/genética , Proteínas Bacterianas/genética , Clonación Molecular , Genes Bacterianos , Glucosa-1-Fosfato Adenililtransferasa/genética , Glucógeno/metabolismo , Cinética , Estructura Cuaternaria de Proteína

RESUMEN

Cell wall degradation by cellulases is extensively explored owing to its potential contribution to biofuel production. The cellulosome is an extracellular multienzyme complex that can degrade the plant cell wall very efficiently, and cellulosomal enzymes are therefore of great interest. The cellulosomal cellulases are defined as enzymes that contain a dockerin module, which can interact with a cohesin module contained in multiple copies in a noncatalytic protein, termed scaffoldin. The assembly of the cellulosomal cellulases into the cellulosomal complex occurs via specific protein-protein interactions. Cellulosome systems have been described initially only in several anaerobic cellulolytic bacteria. However, owing to ongoing genome sequencing and metagenomic projects, the discovery of novel cellulosome-producing bacteria and the description of their cellulosomal genes have dramatically increased in the recent years. In this chapter, methods for discovery of novel cellulosomal cellulases from a DNA sequence by bioinformatics and biochemical tools are described. Their biochemical characterization is also described, including both the enzymatic activity of the putative cellulases and their assembly into mature designer cellulosomes.

Asunto(s)

Bioquímica/métodos , Celulasas/metabolismo , Celulosomas/metabolismo , Genómica/métodos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas de Ciclo Celular/genética , Celulasas/química , Celulosa/metabolismo , Proteínas Cromosómicas no Histona/genética , Biología Computacional , Secuencia Conservada , Genoma Bacteriano , Filogenia , Ruminococcus/enzimología , Ruminococcus/genética , Cohesinas

RESUMEN

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Multimerización de Proteína , Ruminococcus/enzimología , Proteínas Bacterianas/química , Calorimetría , Proteínas Portadoras/química , Celulosomas/metabolismo , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Unión Proteica , Conformación Proteica , Ruminococcus/metabolismo

RESUMEN

Carbohydrate hydrolyzing α-glucosidases are commonly found in microorganisms present in the human intestine microbiome. We have previously reported crystal structures of an α-glucosidase from the human gut bacterium Blaubia (Ruminococcus) obeum (Ro-αG1) and its substrate preference/specificity switch. This novel member of the GH31 family is a structural homolog of human intestinal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) with a highly conserved active site that is predicted to be common in Ro-αG1 homologs among other species that colonize the human gut. In this report, we present structures of Ro-αG1 in complex with the antidiabetic α-glucosidase inhibitors voglibose, miglitol, and acarbose and supporting binding data. The in vitro binding of these antidiabetic drugs to Ro-αG1 suggests the potential for unintended in vivo crossreaction of the α-glucosidase inhibitors to bacterial α-glucosidases that are present in gut microorganism communities. Moreover, analysis of these drug-bound enzyme structures could benefit further antidiabetic drug development.

Asunto(s)

Proteínas Bacterianas/metabolismo , Microbioma Gastrointestinal/fisiología , Inhibidores de Glicósido Hidrolasas/metabolismo , Hipoglucemiantes/metabolismo , alfa-Glucosidasas/metabolismo , 1-Desoxinojirimicina/análogos & derivados , Proteínas Bacterianas/química , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacocinética , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacocinética , Inositol/análogos & derivados , Modelos Moleculares , Unión Proteica , Ruminococcus/enzimología , alfa-Glucosidasas/química

RESUMEN

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , ARN Bacteriano/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Bases de Datos Genéticas , Escherichia coli/enzimología , Escherichia coli/genética , Eubacterium/enzimología , Eubacterium/genética , Regulación Bacteriana de la Expresión Génica , Conformación de Ácido Nucleico , Dominios Proteicos , Estructura Secundaria de Proteína , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Ruminococcus/enzimología , Ruminococcus/genética , Relación Estructura-Actividad

RESUMEN

Ruminococcus gnavus is a human gut symbiont wherein the ability to degrade mucins is mediated by an intramolecular trans-sialidase (RgNanH). RgNanH comprises a GH33 catalytic domain and a sialic acid-binding carbohydrate-binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis and binding assays to determine the structure and function of RgNanH_CBM40 (RgCBM40). RgCBM40 displays the canonical CBM40 ß-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. RgCBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that RgCBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut.

Asunto(s)

Adhesinas Bacterianas/química , Glicoproteínas/química , Mucinas/metabolismo , Ácido N-Acetilneuramínico/química , Neuraminidasa/química , Ruminococcus/enzimología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Dominio Catalítico/genética , Línea Celular , Colon/citología , Colon/metabolismo , Biología Computacional , Cristalografía por Rayos X , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células Caliciformes/metabolismo , Humanos , Lactosa/análogos & derivados , Lactosa/química , Lactosa/metabolismo , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/genética , Neuraminidasa/metabolismo , Unión Proteica , Especificidad por Sustrato , Simbiosis

RESUMEN

Naturally occurring 2,7-anhydro-alpha-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) is a transglycosylation product of bacterial intramolecular trans-sialidases (IT-sialidases). A facile one-pot two-enzyme approach has been established for the synthesis of 2,7-anhydro-sialic acid derivatives including those containing different sialic acid forms such as Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The approach is based on the use of Ruminoccocus gnavus IT-sialidase for the release of 2,7-anhydro-sialic acid from glycoproteins, and the conversion of free sialic acid by a sialic acid aldolase. This synthetic method, which is based on a membrane-enclosed enzymatic synthesis, can be performed on a preparative scale. Using fetuin as a substrate, high-yield and cost-effective production of 2,7-anhydro-Neu5Ac was obtained to high-purity. This method was also applied to the synthesis of 2,7-anhydro-Neu5Gc. The membrane-enclosed multienzyme (MEME) strategy reported here provides an efficient approach to produce a variety of sialic acid derivatives.

Asunto(s)

Glicoproteínas/metabolismo , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismo , Ruminococcus/enzimología , Ruminococcus/metabolismo

RESUMEN

Non-classical protein secretion in bacteria is a common phenomenon. However, the selection principle for non-classical secretion pathways remains unclear. Here, our experimental data, to our knowledge, are the first to show that folded multimeric proteins can be recognized and excreted by a non-classical secretion pathway in Bacillus subtilis. We explored the secretion pattern of a typical cytoplasmic protein D-psicose 3-epimerase from Ruminococcus sp. 5_1_39BFAA (RDPE), and showed that its non-classical secretion is not simply due to cell lysis. Analysis of truncation variants revealed that the C- and N-terminus, and two hydrophobic domains, are required for structural stability and non-classical secretion of RDPE. Alanine scanning mutagenesis of the hydrophobic segments of RDPE revealed that hydrophobic residues mediated the equilibrium between its folded and unfolded forms. Reporter mCherry and GFP fusions with RDPE regions show that its secretion requires an intact tetrameric protein complex. Using cross-linked tetramers, we show that folded tetrameric RDPE can be secreted as a single unit. Finally, we provide evidence that the non-classical secretion pathway has a strong preference for multimeric substrates, which accumulate at the poles and septum region. Altogether, these data show that a multimer recognition mechanism is likely applicable across the non-classical secretion pathway.

Asunto(s)

Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Carbohidrato Epimerasas/metabolismo , Ruminococcus/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Carbohidrato Epimerasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ruminococcus/enzimología