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1.
Nat Commun ; 11(1): 4017, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782292

RESUMO

The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.


Assuntos
Microbioma Gastrointestinal , Hexosaminidases/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Animais , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hexosaminidases/química , Hexosaminidases/genética , Humanos , Glicoproteínas de Membrana/química , Estrutura Molecular , Mucinas/química , Filogenia , Polissacarídeos/química , Polissacarídeos/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
2.
Nat Commun ; 11(1): 3391, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636369

RESUMO

Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3-mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair.


Assuntos
Adenosina Difosfato Ribose/química , Cromatina/química , Quebras de DNA de Cadeia Simples , Reparo do DNA , Glicosídeo Hidrolases/genética , Mutação , Linhagem Celular Tumoral , Sobrevivência Celular , Fibroblastos , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Histonas/química , Humanos , Doenças Neurodegenerativas/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética
3.
PLoS One ; 15(6): e0231513, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479540

RESUMO

Enzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of ß-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of ß-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular ß-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing ß-1,2-linked arabinofuranose disaccharide (ß-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (α/α)6 barrel domain and two flanking (N- and C-terminal) ß-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the ß-Ara2 disaccharide. Three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of structural homologs in other GH families: GH63 α-glycosidase, GH94 chitobiose phosphorylase, GH142 ß-L-arabinofuranosidase, GH78 α-L-rhamnosidase, and GH37 α,α-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. ß-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the ß-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.


Assuntos
Glicosídeo Hidrolases/química , Sequência de Aminoácidos , Bifidobacterium longum/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Alinhamento de Sequência
4.
Nat Commun ; 11(1): 3090, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32555161

RESUMO

Brassicales plants produce glucosinolates and myrosinases that generate toxic isothiocyanates conferring broad resistance against pathogens and herbivorous insects. Nevertheless, some cosmopolitan fungal pathogens, such as the necrotrophic white mold Sclerotinia sclerotiorum, are able to infect many plant hosts including glucosinolate producers. Here, we show that S. sclerotiorum infection activates the glucosinolate-myrosinase system, and isothiocyanates contribute to resistance against this fungus. S. sclerotiorum metabolizes isothiocyanates via two independent pathways: conjugation to glutathione and, more effectively, hydrolysis to amines. The latter pathway features an isothiocyanate hydrolase that is homologous to a previously characterized bacterial enzyme, and converts isothiocyanate into products that are not toxic to the fungus. The isothiocyanate hydrolase promotes fungal growth in the presence of the toxins, and contributes to the virulence of S. sclerotiorum on glucosinolate-producing plants.


Assuntos
Ascomicetos/enzimologia , Ascomicetos/metabolismo , Glucosinolatos/metabolismo , Glicosídeo Hidrolases/metabolismo , Glutationa/metabolismo , Glicosídeo Hidrolases/classificação , Glicosídeo Hidrolases/genética , Hidrólise , Isotiocianatos/metabolismo , Filogenia , Imunidade Vegetal/fisiologia
5.
Nucleic Acids Res ; 48(10): 5656-5669, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329777

RESUMO

Intron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here, we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cell's transcriptional regime.


Assuntos
Acetilglucosamina/metabolismo , Glicosídeo Hidrolases/genética , Íntrons , N-Acetilglucosaminiltransferases/genética , Processamento de RNA , Linhagem Celular , Glicosídeo Hidrolases/metabolismo , Células HEK293 , Humanos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA-Seq
6.
J Biosci Bioeng ; 130(2): 137-141, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32331776

RESUMO

We had developed a new pretreatment system using cow rumen fluid to improve the methane production from lignocellulosic substrates. However, the pretreatment conditions differ from the in-situ rumen environment, therefore different microbes may be involved in plant cell wall decomposition. In the current study, shotgun metagenomic analysis using MiSeq platform was performed to elucidate the bacteria which produce cellulase and hemicellulase in this pretreatment system. The rumen fluid which contained waste paper pieces (0.1% w/v) were incubated at 37°C during 120 h. The fluid samples were collected from the reactor at each time-point and analyzed for chemical properties. Rumen microbial DNA was extracted from 0-h and 60-h samples and subjected to shotgun-metagenomic analysis. After pretreatment, approximately half of cellulose and hemicellulose contents of the waste paper were decomposed and some volatile fatty acids were accumulated. Clostridia (e.g., Ruminococcus and Clostridium) were the predominant bacteria before and after 60-h pretreatment, and their relative abundance was increased during pretreatment. However, Prevotella and Fibrobacter, one of the most dominant bacteria in-situ rumen fluid, were observed less than 3% before incubation and they were decreased after pretreatment. Genes encoding cellulase and hemicellulase were mainly found in Ruminococcus, Clostridium, and Caldicellulosiruptor. Calicellulosiruptor, which had not been previously identified as the predominant genus in lignocellulose decomposition in in-situ rumen conditions, might be considered as the main fibrolytic bacterium in this system. Thus, this study demonstrated that the composition of fibrolytic bacteria in this system was greatly different from those in the in-situ rumen.


Assuntos
Bactérias/classificação , Bactérias/enzimologia , Microbiologia Industrial , Lignina/metabolismo , Metagenoma , Rúmen/microbiologia , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Biodiversidade , Biomassa , Bovinos , Celulase/genética , Celulase/metabolismo , Ácidos Graxos Voláteis , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Metano/biossíntese
7.
Arch Microbiol ; 202(7): 1749-1756, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32335717

RESUMO

ß-glucanases are widely applied in biological control, brewing and feed industries; however, there are seldom studies of ß-glucanases in probiotics. Here, ß-glucanase genes were cloned from Bacillus licheniformis, Lactobacillus fermentum and L. johnsonii. ß-glucanase genes, as blg, lfg and ljg isolated from B. licheniformis, L. fermentum and L. johnsonii were prokaryotic expressed to obtain recombinant strains BL, LF and LJ, respectively. Directed mutations in these genes were introduced by sequential error-prone PCR. Results showed that ß-glucanase activities in three mutants mblg, mlfg and mljg were 1.94-, 2.72- and 1.29-fold higher than the BL, LF and LJ, respectively. Mutation sites analysis showed substitutions at Ser370Gly and Leu395Phe in mblg; Arg169His and Asn302Ser in mlfg; Val132Met, Ser226Asn, and Asp355Gly in mljg. Spatial structural predictions revealed the numbers and positions of α-helices and ß-strands in the three mutants were altered, which might result in ß-glucanase activity increasement. Analysis of ß-glucanase properties revealed no significant differences in the optimal temperatures and pH between mutant and wild-type strains. However, mlfg and mljg exhibited greater thermal stability at 30-50 â„ƒ than the wild-type strains, and mblg improved pH stability compared with wild-type strain. This is the first report about ß-glucanase-encoding genes in L. fermentum and L. johnsonii. These findings provide an efficient way to improve the activity of ß-glucanase.


Assuntos
Bacillus , Estabilidade Enzimática/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Lactobacillus , Probióticos , Bacillus/enzimologia , Bacillus/genética , Clonagem Molecular , Concentração de Íons de Hidrogênio , Lactobacillus/enzimologia , Lactobacillus/genética , Mutação , Reação em Cadeia da Polimerase , Temperatura
8.
Nat Commun ; 11(1): 899, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060313

RESUMO

The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-ß-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine. Here, we provide structural snapshots of EndoBT-3987, including the unliganded form, the EndoBT-3987-Man9GlcNAc2Asn substrate complex, and two EndoBT-3987-Man9GlcNAc and EndoBT-3987-Man5GlcNAc product complexes. In combination with alanine scanning mutagenesis and activity measurements we unveil the molecular mechanism of HM-type recognition and specificity for EndoBT-3987 and an important group of the GH18 ENGases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.


Assuntos
Bacteroides thetaiotaomicron/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides thetaiotaomicron/química , Bacteroides thetaiotaomicron/enzimologia , Bacteroides thetaiotaomicron/genética , Microbioma Gastrointestinal , Regulação Bacteriana da Expressão Gênica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Manose/química , Manose/metabolismo , Especificidade por Substrato
9.
Arch Microbiol ; 202(5): 1117-1126, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32060600

RESUMO

Gayal (Bos frontalis) of the Yunnan region is well adapted to harsh environmental conditions. Its diet consists predominantly of bamboo, reeds, and woody plants, suggesting that the rumen of this species contains many fiber-degrading bacteria and cellulases. The aim of this study was to identify and modify specific cellulases found in the gayal rumen. In the present study, a directed evolution strategy of error-prone PCR was employed to improve the activity or optimal temperature of a cellulase gene (CMC-1) isolated from gayal rumen. The CMC-1 gene was heterologously expressed in Escherichia coli (E. coli) BL21, and the recombinant CMC-1 protein hydrolyzed carboxyl methyl cellulose (CMC) with an optimal activity at pH 5.0 and 50 °C. A library of mutated ruminal CMC-1 genes was constructed and a mutant EP-15 gene was identified. Sequencing analysis revealed that EP-15 and CMC-1 belonged to the glycosyl hydrolase family 5 (GHF5) and had the highest homology to a cellulase (Accession No. WP_083429257.1) from Prevotellaceae bacterium, HUN156. There were similar predicted GH5 domains in EP-15 and CMC-1. The EP-15 gene was heterologously expressed and exhibited cellulase activity in E. coli BL21 at pH 5.0, but the optimum temperature for its activity was reduced from that of CMC-1 (50 °C) to 45 °C, which was closer to the physiological temperature of the rumen (40 °C). The cellulase activity of EP-15 was about two times higher than CMC-1 at 45 °C or PH 5.0, and also was more stable in response to temperature and pH changes compared to CMC-1. This study successfully isolated and modified a ruminal cellulase gene from metagenomics library of Yunnan gayal. Our findings may obtain a useful cellulase in future applications and present the first evidence of modified cellulases in the gayal rumen.


Assuntos
Bactérias/genética , Carboximetilcelulose Sódica/metabolismo , Celulases/genética , Glicosídeo Hidrolases/genética , Rúmen/microbiologia , Animais , Bovinos , Celulases/metabolismo , China , Clonagem Molecular , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Metagenoma , Metagenômica , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
10.
Appl Microbiol Biotechnol ; 104(5): 1883-1890, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932892

RESUMO

C-Glycosides, a special type of glycoside, are frequently distributed in many kinds of medicinal plants, such as puerarin and mangiferin, showing various and significant bioactivities. C-Glycosides are usually characterized by the C-C bond that forms between the anomeric carbon of sugar moieties and the carbon atom of aglycon, which is usually resistant against acidic hydrolysis and enzymatic treatments. Interestingly, C-glycosides could be cleaved by several intestinal bacteria, but whether the enzymatic cleavage of C-C glycosidic bond is reduction or hydrolysis has been controversial; furthermore, whether existence of a "C-glycosidase" directly catalyzing the cleavage is not clear. Here we review research advances about the discovery and mechanism of intestinal bacteria in enzymatic cleavage of C-C glycosidic bond with an emphasis on the identification of enzymes manipulation the deglycosylation. Finally, we give a brief conclusion about the mechanism of C-glycoside deglycosylation and perspectives for future study in this field.


Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/metabolismo , Intestinos/microbiologia , Animais , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Biotransformação , Glicosídeo Hidrolases/genética , Glicosídeos/química , Glicosilação , Humanos , Estrutura Molecular
11.
Appl Microbiol Biotechnol ; 104(5): 2079-2096, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31980921

RESUMO

PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the ß-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Enzimas Multifuncionais/metabolismo , Paenibacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Celulase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Hidrólise , Cinética , Manosidases/metabolismo , Simulação de Acoplamento Molecular , Enzimas Multifuncionais/química , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/isolamento & purificação , Mutação , Oligossacarídeos/biossíntese , Paenibacillus/genética , Paenibacillus/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Xilosidases/metabolismo
12.
J Microbiol Biotechnol ; 30(3): 391-397, 2020 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-31893597

RESUMO

In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenoside-converting Blastococcus saxobsidens that was cloned and expressed in Escherichia coli BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb2 into Rd. The gene, termed AbpBs, consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb2 under optimal conditions (pH 7.0 and 40°;C). Kinetic parameters for α-Larabinopyranosidase showed apparent Km and Vmax values of 0.078 ± 0.0002 micrometer and 1.4 ± 0.1 µmol/min/mg of protein against p-nitrophenyl-α-L-arabinopyranoside. Using a purified AbpBs (1 µg/ml), 0.1% of ginsenoside Rb2 was completely converted to ginsenoside Rd within 1 h. The recombinant AbpBs could be useful for high-yield, rapid, and low-cost preparation of ginsenoside Rd from Rb2.


Assuntos
Actinobacteria/enzimologia , Ginsenosídeos/metabolismo , Glicosídeo Hidrolases/metabolismo , Arabinonucleosídeos/metabolismo , Clonagem Molecular , Ginsenosídeos/química , Ginsenosídeos/genética , Glicosídeo Hidrolases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Sci Rep ; 10(1): 1329, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992772

RESUMO

The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: ß-N-acetyl-glucosaminidases, ß-1,4-glucosidases/ß-xylosidases and macrolide ß-glucosidases. The RmNag3 with additional ß-lactamase domain clustered with the deepest rooted GH3-lineage of ß-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed ß-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide ß-glucosidases from Actinomycetes. The ß-xylosidases, RmXyl3A and RmXyl3B, and the ß-glucosidases RmBgl3A and RmBgl3C clustered within the major ß-glucosidases/ß-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed ß-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed ß-1,4-glucosidase/ß-xylosidase activity while RmBgl3C was active on pNP-ß-Glc and ß-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252T Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Família Multigênica , Rhodothermus/enzimologia , Rhodothermus/genética , Ativação Enzimática , Ordem dos Genes , Genes Bacterianos , Loci Gênicos , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/classificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Temperatura
15.
Arterioscler Thromb Vasc Biol ; 40(3): 583-596, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31996026

RESUMO

OBJECTIVE: Inflammatory activation changes the mitochondrial function of macrophages from oxidative phosphorylation to reactive oxygen species production, which may promote necrotic core formation in atherosclerotic lesions. In hypoxic and cancer cells, HIF-1α (hypoxia-inducible factor) promotes oxygen-independent energy production by microRNAs. Therefore, we studied the role of HIF-1α in the regulation of macrophage energy metabolism in the context of atherosclerosis. Approach and Results: Myeloid cell-specific deletion of Hif1a reduced atherosclerosis and necrotic core formation by limiting macrophage necroptosis in apolipoprotein E-deficient mice. In inflammatory bone marrow-derived macrophages, deletion of Hif1a increased oxidative phosphorylation, ATP levels, and the expression of genes encoding mitochondrial proteins and reduced reactive oxygen species production and necroptosis. microRNA expression profiling showed that HIF-1α upregulates miR-210 and downregulates miR-383 levels in lesional macrophages and inflammatory bone marrow-derived macrophages. In contrast to miR-210, which inhibited oxidative phosphorylation and enhanced mitochondrial reactive oxygen species production, miR-383 increased ATP levels and inhibited necroptosis. The effect of miR-210 was due to targeting 2,4-dienoyl-CoA reductase, which is essential in the ß oxidation of unsaturated fatty acids. miR-383 affected the DNA damage repair pathway in bone marrow-derived macrophages by targeting poly(ADP-ribose)-glycohydrolase (Parg), which reduced energy consumption and increased cell survival. Blocking the targeting of Parg by miR-383 prevented the protective effect of Hif1a deletion in macrophages on atherosclerosis and necrotic core formation in mice. CONCLUSIONS: Our findings unveil a new mechanism by which activation of HIF-1α in inflammatory macrophages increases necroptosis through microRNA-mediated ATP depletion, thus increasing atherosclerosis by necrotic core formation.


Assuntos
Aorta/metabolismo , Aterosclerose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Necroptose , Trifosfato de Adenosina/metabolismo , Animais , Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Modelos Animais de Doenças , Metabolismo Energético , Regulação da Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamação/genética , Inflamação/patologia , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , MicroRNAs/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
16.
Artigo em Inglês | MEDLINE | ID: mdl-31647988

RESUMO

This review discusses the reaction catalysed, and the structure and function of the cellulase, endo-ß-1,4-glucanase and the hemicellulase enzymes, ß-1,3-glucanase and endo-ß-1,4-mannase that are present in numerous invertebrate groups with a diverse range of feeding specialisations. These range from microbial deposit and filter feeders, micro and macrophagous algal feeders, omnivores to herbivorous leaf litter and wood feeders. Endo-ß-1,4-glucanase from glycosyl hydrolase family 9 (GH9) digests cellulose like ß-1,4-glucans from a range of materials. As it hydrolyses crystalline cellulose very slowly, it is a poor cellulase. Where tested, the enzyme has dual endo-ß-1,4-glucanase and lichenase activity. Its presence does not necessarily indicate the ability of an animal to digest cellulose. It only indicates the ability to digest ß-1,4-glucans and its function, which is discussed in this review, should be considered with reference to the substrates present in the diet. ß-1,3-glucanase (laminarinase) belongs to glycosyl hydrolase family 16 (GH16) and hydrolyses ß-1.3-glucans. These polysaccharides are present in the cell walls of algae, protozoans and yeast, and they also occur as storage polysaccharides within protozoans and algae. Depending on their site of expression, these enzymes may function as a digestive enzyme or may be involved in innate immunity. Enzymes present in the digestive fluids or tissues, would be digestive. Haemolymph GH16 proteins may be involved in innate immunity through the activation of the phenol oxidase system. Insect GH16 proteins expressed within the haemolymph have lost their catalytic residues and function as ß-glucan binding proteins. In contrast, crustacean GH16 proteins expressed within the same tissue, have retained the catalytic residues and thus possibly their ß-1,3-glucanase activity. The potential function of which is discussed. Endo-ß-1,4-mannase from glycosyl hydrolase family 5, subfamily 10 (GH5_10) hydrolyses mannan, glucomannan and galactomannan. These hemicelluloses are present in the cell walls of plants and algae and also function as storage polysaccharides within legume and palm seeds. They are digestive enzymes whose high expression in some species suggests they are a major contributor to hemicellulose digestion. They may also provide the animal with substantial amounts of monosaccharides for energy.


Assuntos
Proteínas de Artrópodes , Celulase , Glicosídeo Hidrolases , Invertebrados , Filogenia , Polissacarídeos/metabolismo , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Celulase/genética , Celulase/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Invertebrados/enzimologia , Invertebrados/genética
17.
Enzyme Microb Technol ; 133: 109447, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874680

RESUMO

To increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzymes acting cooperatively, including endoxylanases (XYN), ß-xylosidases (XYL) and α-l-arabinofuranosidases (ABF) is necessary. The current work aimed at evaluating the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of steam-exploded sugarcane bagasse (SEB). One recombinant endoxylanase (HXYN2) and one recombinant ß-xylosidase (HXYLA) from Humicola grisea var thermoidea, together with an α-l-arabinofuranosidase (AFB3) from Penicillium pupurogenum, all produced in Pichia pastoris, were used to formulate an efficient enzyme mixture for SEB hydrolysis using a 23 Central Composite Rotatable Design (CCRD). The most potent enzyme for SEB hydrolysis was ABF3. Subsequently, the optimal enzyme mixture was used in combination with commercial cellulases (Accellerase 1500), either simultaneously or in sequential experiments. The supplementation of Accellerase 1500 with hemicellulases enhanced the glucose yield from SEB hydrolysis by 14.6%, but this effect could be raised to 50% when hemicellulases were added prior to hydrolysis with commercial cellulases. These results were supported by scanning electron microscopy, which revealed the effect of enzymatic hydrolysis on SEB fibers. Our results show the potential of complementary enzyme activities to improve enzymatic hydrolysis of SEB, thus improving the efficiency of the hydrolytic process.


Assuntos
Celulose , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Saccharum/metabolismo , Vapor , Celulose/metabolismo , Hidrólise , Penicillium/enzimologia , Penicillium/genética , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Int J Food Microbiol ; 316: 108476, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-31874325

RESUMO

This work aimed to investigate the ability of two human-derived bifidobacterial strains, i.e. Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809, to utilize various oligosaccharides (i.e., 4-galactosyl-kojibiose, lactulosucrose, lactosyl-oligofructosides, raffinosyl-oligofructosides and lactulose-derived galacto-oligosaccharides) synthesized by means of microbial glycoside hydrolases. With the exception of raffinosyl-oligofructosides, these biosynthetic oligosaccharides were shown to support growth acting as a sole carbon and energy source of at least one of the two studied strains. Production of short-chain fatty acids (SCFAs) as detected by HPLC analysis corroborated the suitability of most of the studied novel oligosaccharides as fermentable growth substrates for the two bifidobacterial strains, showing that acetic acid is the main metabolic end product followed by lactic and formic acids. Transcriptomic and functional genomic approaches carried out for B. breve UCC2003 allowed the identification of key genes encoding glycoside hydrolases and carbohydrate transport systems involved in the metabolism of 4-galactosyl-kojibiose and lactulosucrose. In particular, the role of ß-galactosidases in the hydrolysis of these particular trisaccharides was demonstrated, highlighting their importance in oligosaccharide metabolism by human bifidobacterial strains.


Assuntos
Bifidobacterium breve/metabolismo , Bifidobacterium longum/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Bactérias/genética , Bifidobacterium breve/crescimento & desenvolvimento , Bifidobacterium breve/isolamento & purificação , Bifidobacterium longum/crescimento & desenvolvimento , Bifidobacterium longum/isolamento & purificação , Metabolismo dos Carboidratos/genética , Ácidos Graxos Voláteis/biossíntese , Ácidos Graxos Voláteis/química , Fermentação , Glicosídeo Hidrolases/genética , Humanos , Oligossacarídeos/química , Transcriptoma , beta-Galactosidase/genética
19.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140294, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676454

RESUMO

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and ß-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Šfrom the active site, the single mutants had 15-40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for ß-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-63-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51-109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and ß-limit dextrin.


Assuntos
Glicosídeo Hidrolases/química , Hordeum/enzimologia , Proteínas de Plantas/química , Amilopectina/química , Sítios de Ligação , Catálise , Dextrinas/química , Glucanos/química , Glicosídeo Hidrolases/genética , Modelos Moleculares , Pichia/genética , Proteínas de Plantas/genética , Domínios Proteicos , Proteínas Recombinantes/química , Especificidade por Substrato
20.
Protein Expr Purif ; 166: 105519, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31629955

RESUMO

Chitosanase (EC 3.2.1.132) is an important chitosan-degrading enzyme involved in industrial applications. In this study, a chitosanase gene (BbCSN-1) from Beauveria bassiana, an insect fungal pathogen, was cloned and expressed in Pichia pastoris. The amount of BbCSN-1 in the fermentation broth of P. pastoris gradually increased after induction with methanol from one to 6 d, reaching 398 µg/ml on the 6th day. The molecular characteristics of BbCSN-1 were measured with colloidal chitosan as a substrate. The purified BbCSN-1 exhibited optimum activity at pH 5 and 30 °C and was stable at pH 2-8 and below 40 °C. The Km value of BbCSN-1 was approximately 0.8 mg/ml at 30 °C (pH 6.0). The activity of BbCSN-1 was significantly enhanced by Mn2+ but inhibited by Co2+ and Cu2+. These results indicated that BbCSN-1 from B. bassiana could be easily expressed in P. pastoris, which provided a basis for further study on its application.


Assuntos
Beauveria/genética , Glicosídeo Hidrolases/genética , Pichia/genética , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Cátions Bivalentes/química , Clonagem Molecular , Cobalto/química , Cobre/química , Expressão Gênica , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Manganês/química , Pichia/enzimologia , Ligação Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Temperatura , Termodinâmica
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