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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.
Science ; 369(6507): 1077-1084, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855333

RESUMO

Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses.


Assuntos
Adenosina Desaminase/química , Archaea/virologia , Vírus de Archaea/imunologia , Bactérias/virologia , Bacteriófagos/imunologia , Sistemas CRISPR-Cas , Edição de RNA , Adenosina Desaminase/classificação , Adenosina Desaminase/genética , Archaea/enzimologia , Proteínas Arqueais , Bactérias/enzimologia , Proteínas de Bactérias , Genes Arqueais , Genes Bacterianos , Domínios Proteicos
3.
Food Chem ; 332: 127417, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32629332

RESUMO

The use of UHPH sterilization in the absence of SO2 has been used to eliminate wild microorganisms and inactivate oxidative enzymes. A white must of the Muscat of Alexandria grape variety was continuously processed by UHPH at 300 MPa (inlet temperature: 23-25 °C). The initial microbial load of the settled must was 4-log CFU/mL for both yeast and moulds, and slightly lower for bacteria. After UHPH processing, no microorganisms were detected in 1 mL. UHPH musts remain without fermentative activity for more than 60 days. Concentrations of the thermal markers indicated the absence of thermal damage in the UHPH-treated musts, since 5-hydroxymethylfurfural was not detected. In addition, the must treated by UHPH keeps terpene concentrations similar to those of the untreated controls. A strong inactivation of the oxidative enzymes was observed, with no browning at room temperature for more than 3 days. The antioxidant value of the UHPH-treated must was 156% higher than the control.


Assuntos
Bactérias/crescimento & desenvolvimento , Microbiologia de Alimentos/métodos , Fungos/crescimento & desenvolvimento , Esterilização/métodos , Vinho/análise , Bactérias/enzimologia , Bactérias/metabolismo , Fermentação , Manipulação de Alimentos , Fungos/enzimologia , Fungos/genética , Fungos/metabolismo , Humanos , Dióxido de Enxofre/análise , Paladar , Temperatura , Vitis/química
4.
Nucleic Acids Res ; 48(14): 7914-7923, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652039

RESUMO

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter -10 element, while not affecting interactions with -35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of -10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with -10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.


Assuntos
Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Regiões Promotoras Genéticas , Tiofenos/farmacologia , Iniciação da Transcrição Genética/efeitos dos fármacos , Antibacterianos/química , Bactérias/enzimologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fator sigma/antagonistas & inibidores , Fator sigma/química , Tiofenos/química
5.
Proc Natl Acad Sci U S A ; 117(27): 15599-15608, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571930

RESUMO

2(S)-dihydroxypropanesulfonate (DHPS) is a microbial degradation product of 6-deoxy-6-sulfo-d-glucopyranose (sulfoquinovose), a component of plant sulfolipid with an estimated annual production of 1010 tons. DHPS is also at millimolar levels in highly abundant marine phytoplankton. Its degradation and sulfur recycling by microbes, thus, play important roles in the biogeochemical sulfur cycle. However, DHPS degradative pathways in the anaerobic biosphere are not well understood. Here, we report the discovery and characterization of two O2-sensitive glycyl radical enzymes that use distinct mechanisms for DHPS degradation. DHPS-sulfolyase (HpsG) in sulfate- and sulfite-reducing bacteria catalyzes C-S cleavage to release sulfite for use as a terminal electron acceptor in respiration, producing H2S. DHPS-dehydratase (HpfG), in fermenting bacteria, catalyzes C-O cleavage to generate 3-sulfopropionaldehyde, subsequently reduced by the NADH-dependent sulfopropionaldehyde reductase (HpfD). Both enzymes are present in bacteria from diverse environments including human gut, suggesting the contribution of enzymatic radical chemistry to sulfur flux in various anaerobic niches.


Assuntos
Alcanossulfonatos/metabolismo , Anaerobiose , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Microbioma Gastrointestinal/fisiologia , Biologia Computacional , Ensaios Enzimáticos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/toxicidade , Metilglucosídeos/metabolismo , Enxofre/metabolismo
6.
World J Microbiol Biotechnol ; 36(5): 73, 2020 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-32385754

RESUMO

Liometopum apiculatum is a species of ants widely distributed in arid and semi-arid ecosystems where there is a relative food shortage compared with tropical ecosystems. L. apiculatum has established an ecological balance involving symbiotic interactions, which have allowed them to survive through mechanisms that are still unknown. Therefore, the aim of this study was to explore the metabolic potential of isolated bacteria from L. apiculatum using enzymatic activity assay and substrate assimilation. Results revealed a complex bacteria consortium belonging to Proteobacteria, Firmicutes, and Actinobacteria phylum. Most of the isolated bacteria showed activities associated with biopolymers degradation, from them Exiguobacterium and B. simplex showed the highest amylolytic activity (27 U/mg protein), while A. johnsonii and B. pumulis showed the highest cellulolytic and xylanolytic activities (1 and 2.9 U/mg protein, respectively). By other hand, some microorganisms such as S. ficaria, E. asburiae, P. agglomerans, A. johnsonii, S. rubidaea, S. marcescens, S. warneri, and M. hydrocarbonoxydans were able to grow up to 1000 mg/L of phthalates esters. These results not only revealed the important contribution of the symbionts in L apiculatum ants feeding habits, but also have shown a promising source of enzymes with potential biotechnological applications such as lignocellulosic biomass hydrolysis and bioremediation processes.


Assuntos
Formigas/microbiologia , Bactérias/isolamento & purificação , Bactérias/metabolismo , Biodegradação Ambiental , Microbiota/fisiologia , Animais , Bactérias/classificação , Bactérias/enzimologia , Biomassa , Celulose/metabolismo , Hábitos , Hidrólise , Larva/microbiologia , Lignina/metabolismo , Polissacarídeos/metabolismo , Simbiose , Xilanos/metabolismo
7.
Med Sci (Paris) ; 36(5): 465-471, 2020 May.
Artigo em Francês | MEDLINE | ID: mdl-32452368

RESUMO

In the last decade, the association between the periodontitis and rheumatoid arthritis (RA) has been established, suggesting that oral microbiome plays a causal role by initiating this chronic autoimmune inflammatory disease of articulation. Both pathogenesis are similar in term of chronic inflammation, tissue breakdown and bone resorption. Molecular aspects have also revealed that citrullination, a post-translational modification catalyzed by peptidyl-arginine deiminases (PADs), is involved in both diseases. For RA, citrullinated proteins production leads to the synthesis the of anti-citrullinated protein antibodies triggering the loss of immune tolerance. In humans, five PADs have been identified. Recently, studies have found that only Porphyromonas species possess PAD. Thus, a major periodontal pathogen, Porphyromonas gingivalis, is able to generate citrullinated epitopes, and could consequently induce anti-citrullinated protein antibodies. In this review, citrullination process, periodontitis and RA are described to put them in relation with molecular, clinical and epidemiological studies establishing the association between periodontitis and RA.


Assuntos
Artrite Reumatoide/etiologia , Bactérias/enzimologia , Citrulinação/fisiologia , Microbiota/fisiologia , Boca/microbiologia , Desiminases de Arginina em Proteínas/fisiologia , Artrite Reumatoide/enzimologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/microbiologia , Bactérias/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Periodontite/complicações , Periodontite/enzimologia , Periodontite/microbiologia , Processamento de Proteína Pós-Traducional
8.
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
9.
Nucleic Acids Res ; 48(11): 6149-6156, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32347937

RESUMO

Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ribonucleases/metabolismo , Sítios de Ligação , Modelos Moleculares , Domínios Proteicos , RNA/metabolismo , Estabilidade de RNA , Sistemas do Segundo Mensageiro
10.
PLoS One ; 15(4): e0231198, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343698

RESUMO

Soil physicochemical properties, bacterial communities and enzyme activities change with land subsidence resulting from coal mining. However, research on the responses of bacterial communities and enzyme activities to the soil properties in different degree of subsidence areas is limited. As such, we collected soil samples from a control area (C area), a moderate mining subsidence area (M area) and a severe mining subsidence area (S area) in Central China. Soil properties, such as the pH, total nitrogen (TN) content, total phosphorus (TP) content, available phosphorus (AP) content, organic matter (OM) content, and soil enzyme (urease, invertase, catalase and alkaline phosphatase) activities were measured in each sampling area at depths of 0-20 cm, 20-40 cm, and 40-60 cm. The results indicated that the soil physiochemical properties, soil urease activity, soil alkaline phosphatase activity and soil bacterial richness and diversity in the topsoil (0-20 cm) of the mining subsidence area were significantly lower than those in the C area. However, the soil enzyme activities within the deepest layer of the subsidence area were significantly greater than those of the C area. The bacterial communities within the depth of 0-20 cm were dominated by RB41, Pseudomonas, MND1, Nitrospira, Trichococcus, Sphingomonas and Dongia, whereas RB41 and Pseudomonas were the dominant species in the C area and subsidence area, respectively. Using correlation analysis, we found that the soil pH value, soil AP content and activities of the four enzymes were the main factors affecting the soil bacterial community structure. In addition, the soil nutrient contents, enzyme activities and bacterial richness and evenness decreased with increasing subsidence degree (classified by geological hazards, groundwater and landscape damage degree of coal mining subsidence). These results provide a reliable basis for environmental management of mining areas.


Assuntos
Bactérias/enzimologia , Minas de Carvão , Microbiologia do Solo , Solo/química , Fosfatase Alcalina/análise , Catalase/análise , China , Geografia , Água Subterrânea , Concentração de Íons de Hidrogênio , Nitrogênio/análise , Fósforo/análise , Urease/análise , beta-Frutofuranosidase/análise
11.
PLoS One ; 15(4): e0230615, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251430

RESUMO

Availability of good quality irrigation water is a big challenge in arid and semi arid regions of the world. Drought stress results in poor plant growth and low yield; however, the rhizobacteria, capable of producing 1-aminocyclopropane-1-carboxylate (ACC)-deaminase are likely to improve crop growth and productivity under drought stress. Similarly, biochar could also ameliorate the negative impacts of drought stress. Therefore, this pot experiment was conducted to evaluate the role of ACC-deaminase producing plant growth promoting rhizobacteria (PGPR) alone and in combinations with timber-waste biochar in improving maize growth under drought stress. The ACC-deaminase producing rhizobacteria, Pseudomonas aeruginosa, Enterobacter cloacae, Achromobacter xylosoxidans and Leclercia adecarboxylata were studied along with two rates (0.75 and 1.50% of the soil weight) of biochar under three moisture levels i.e., normal moisture, mild drought stress and severe drought stress. The E. cloacae in conjunction with higher rate of biochar produced a significant improvement i.e., up to 60, 73, 43, 69, 76 and 42% respectively, in grain yield plant-1, photosynthetic rate, stomatal conductance, chlorophyll a, total chlorophyll and carotenoids contents of maize as compared to the control under mild drought stress. Similarly, A. xylosoxidans with higher rate of biochar also enhanced grain yield plant-1, photosynthetic rate, stomatal conductance, chlorophyll a, total chlorophyll and carotenoids contents of maize up to 200, 213, 113, 152, 148 and 284%, respectively over control under severe drought stress. In conclusion, combination of ACC-deaminase containing PGPR, A. xylosoxidans and biochar (0.75%) proved an effective technique to improve maize growth and productivity under drought stress.


Assuntos
Bactérias/enzimologia , Carbono-Carbono Liases/biossíntese , Carvão Vegetal/farmacologia , Secas , Rizosfera , Zea mays/efeitos dos fármacos , Zea mays/microbiologia , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Carotenoides/metabolismo , Clorofila/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Prolina/metabolismo , Estresse Fisiológico , Zea mays/crescimento & desenvolvimento , Zea mays/fisiologia
12.
Nat Commun ; 11(1): 1614, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32235841

RESUMO

The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich ring system remains unknown. Here, we report the biosynthetic route to the triazole-bearing antimetabolite 8-azaguanine. We reveal that its triazole moiety can be assembled through an enzymatic and non-enzymatic cascade, in which nitric oxide is used as a building block. These results expand our knowledge of the physiological role of nitric oxide synthase in building natural products with a nitrogen-nitrogen bond, and should also inspire the development of synthetic biology approaches for triazole production.


Assuntos
Bactérias/metabolismo , Óxido Nítrico/metabolismo , Triazóis/metabolismo , Azaguanina/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos Biológicos , Vias Biossintéticas/genética , Genes Bacterianos/genética , Óxido Nítrico Sintase/metabolismo , Nitrogênio , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo , Biologia Sintética
13.
J Nat Med ; 74(3): 501-512, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32180104

RESUMO

Aromatic prenyltransferases (PTases), including ABBA-type and dimethylallyl tryptophan synthase (DMATS)-type enzymes from bacteria and fungi, play important role for diversification of the natural products and improvement of the biological activities. For a decade, the characterization of enzymes and enzymatic synthesis of prenylated compounds by using ABBA-type and DMATS-type PTases have been demonstrated. Here, I introduce several examples of the studies on chemoenzymatic synthesis of unnatural prenylated compounds and the enzyme engineering of ABBA-type and DMATS-type PTases.


Assuntos
Alquil e Aril Transferases/metabolismo , Bactérias/enzimologia , Dimetilaliltranstransferase/metabolismo , Fungos/enzimologia , Engenharia de Proteínas , Bactérias/metabolismo , Produtos Biológicos/metabolismo , Fungos/metabolismo , Prenilação/fisiologia
14.
World J Microbiol Biotechnol ; 36(3): 51, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157408

RESUMO

Culture-independent molecular-based approaches can be used to identify genes of interest from environmental sources that have desirable properties such as thermo activity. For this study, a putative thermo stable endoglucanase gene was identified from a mixed culture resulting from the inoculation of Brock-CMcellulose (1%) broth with mudspring water from Mt. Makiling, Laguna, Philippines that had been incubated at 90 °C. Genomic DNA was extracted from the cellulose-enriched mixed culture and endo1949 forward and reverse primers were used to amplify the endoglucanase gene, which was cloned into pCR-script plasmid vector. Blastn alignment of the sequenced insert revealed 99.69% similarity to the glycosyl hydrolase, sso1354 (CelA1; Q97YG7) from Saccharolobus solfataricus. The endoglucanase gene (GenBank accession number MK984682) was determined to be 1,021 nucleotide bases in length, corresponding to 333 amino acids with a molecular mass of ~ 37 kDa. The endoglucanase gene was inserted into a pET21 vector and transformed in E. coli BL21 for expression. Partially purified recombinant Mt. Makiling endoglucanase (MM-Engl) showed a specific activity of 187.61 U/mg and demonstrated heat stability up to 80 °C. The thermo-acid stable endoglucanase can be used in a supplementary hydrolysis step to further hydrolyze the lignocellulosic materials that were previously treated under high temperature-dilute acid conditions, thereby enhancing the release of more glucose sugars for bioethanol production.


Assuntos
Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , DNA , Genômica , Água/metabolismo , Sequência de Aminoácidos , Archaea/enzimologia , Archaea/genética , Bactérias/enzimologia , Bactérias/genética , Sequência de Bases , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Filipinas , Proteínas Recombinantes , Alinhamento de Sequência , Sulfolobales/enzimologia , Sulfolobales/genética , Temperatura , Microbiologia da Água
15.
World J Microbiol Biotechnol ; 36(4): 53, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32172335

RESUMO

The recent scientific progresses on the use of enzyme-mediated reactions in organic, non-aqueous and aqueous media have significantly supported the growing demand of new biotechnological and/or pharmacological products. Today, a plethora of microbial enzymes, used as biocatalysts, are available. Among these, microbial transglutaminases (MTGs) are broadly used for their ability to catalyse the formation of an isopeptide bond between the γ-amide group of glutamines and the ε-amino group of lysine. Due to their promiscuity towards primary amine-containing substrates and the more stringent specificity for glutamine-containing peptide sequences, several combined approaches can be tailored for different settings, making MTGs very attractive catalysts for generating protein-protein and protein small molecule's conjugates. The present review offers a recent update on the modifications attainable by MTG-catalysed bioreactions as reported between 2014 and 2019. In particular, we present a detailed and comparative overview on the MTG-based methods for proteins and antibodies engineering, with a particular outlook on the synthesis of homogeneous antibody-drug conjugates.


Assuntos
Bactérias/enzimologia , Fungos/enzimologia , Engenharia de Proteínas/métodos , Transglutaminases/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Proteínas Fúngicas/metabolismo , Imunoconjugados/metabolismo , Especificidade por Substrato
16.
Arterioscler Thromb Vasc Biol ; 40(5): 1239-1255, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32212854

RESUMO

OBJECTIVE: Gut microbial metabolism of dietary choline, a nutrient abundant in a Western diet, produces trimethylamine (TMA) and the atherothrombosis- and fibrosis-promoting metabolite TMA-N-oxide (TMAO). Recent clinical and animal studies reveal that elevated TMAO levels are associated with heightened risks for both cardiovascular disease and incident chronic kidney disease development. Despite this, studies focusing on therapeutically targeting gut microbiota-dependent TMAO production and its impact on preserving renal function are limited. Approach and Results: Herein we examined the impact of pharmacological inhibition of choline diet-induced gut microbiota-dependent production of TMA, and consequently TMAO, on renal tubulointerstitial fibrosis and functional impairment in a model of chronic kidney disease. Initial studies with a gut microbial choline TMA-lyase mechanism-based inhibitor, iodomethylcholine, confirmed both marked suppression of TMA generation, and consequently TMAO levels, and selective targeting of the gut microbial compartment (ie, both accumulation of the drug in intestinal microbes and limited systemic exposure in the host). Dietary supplementation of either choline or TMAO significantly augmented multiple indices of renal functional impairment and fibrosis associated with chronic subcutaneous infusion of isoproterenol. However, the presence of the gut microbiota-targeting inhibitor iodomethylcholine blocked choline diet-induced elevation in TMAO, and both significantly improved decline in renal function, and significantly attenuated multiple indices of tubulointerstitial fibrosis. Iodomethylcholine treatment also reversed many choline diet-induced changes in cecal microbial community composition associated with TMAO and renal functional impairment. CONCLUSIONS: Selective targeting of gut microbiota-dependent TMAO generation may prevent adverse renal structural and functional alterations in subjects at risk for chronic kidney disease.


Assuntos
Bactérias/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Colina/farmacologia , Inibidores Enzimáticos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Rim/efeitos dos fármacos , Liases/antagonistas & inibidores , Metilaminas/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Animais , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Colina/análogos & derivados , Modelos Animais de Doenças , Fibrose , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Liases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/microbiologia , Insuficiência Renal Crônica/patologia
17.
Arch Biochem Biophys ; 685: 108350, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32220566

RESUMO

Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/química , Hidrólise , Mutagênese Sítio-Dirigida , Mutação , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Potássio/metabolismo , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato
18.
Nat Chem Biol ; 16(3): 318-326, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32042200

RESUMO

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.


Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Microbioma Gastrointestinal/fisiologia , Amidoidrolases/fisiologia , Animais , Bactérias/enzimologia , Ácidos e Sais Biliares/metabolismo , Desenho de Fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bibliotecas de Moléculas Pequenas
19.
Artigo em Inglês | MEDLINE | ID: mdl-32069792

RESUMO

Hospital sewage plays a key role in the dissemination of antibiotic-resistant genes (ARGs) by serving as an environmental antimicrobial resistance reservoir. In this study, we aimed to characterize the cephalosporin- and carbapenem-resistant isolates from hospital sewage and receiving rivers. The results showed that ESBL (blaCTX-M) and carbapenemase genes (blaNDM and blaKPC) were widely detected in a number of different bacterial species. These resistance genes were mainly harbored in Enterobacteriaceae, followed by Acinetobacter and Aeromonas isolates. More attention should be given to these bacteria as important vectors of ARGs in the environment. Furthermore, we showed that the multidrug resistance phenotype was highly prevalent, which was found in 85.5% Enterobacteriaceae and 75% Acinetobacter strains. Notably, the presence of carbapenemase genes in isolates from treated effluents and receiving rivers indicates that the discharges of wastewater treatment plants could be an important source for high-risk resistance genes propagation to the environment. In conclusion, this study shows a high prevalence of ESBL- and carbapenemase-producing bacteria in hospital sewage and receiving rivers in China. These findings have serious implications for human health, and also suggest the need for more efforts to control the dissemination of resistant bacteria from hospital sewage into the environment.


Assuntos
Bactérias , Proteínas de Bactérias , Esgotos , beta-Lactamases , Antibacterianos , Bactérias/enzimologia , Bactérias/metabolismo , China , Humanos , Testes de Sensibilidade Microbiana , Prevalência , Rios , Esgotos/microbiologia
20.
Ecotoxicol Environ Saf ; 192: 110294, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32044601

RESUMO

Cadmium (Cd) and nickel (Ni) in soil have caused serious environmental problems and increased healthy risks to humans and biota, it is vital important and necessary to develop effective methods to resolve the combined contaminated problems. In this study, strains L5 and L6 with good heavy metal resistant and immobilizing capacities were isolated from Cd and Ni contaminated soil. Bacterial characteristic experiment illustrated that many functional groups (-OH, -NH2 and -COO et al.) were distributed on the surface of L5 and L6. Under the stress of heavy metals, bacterial appearances were distorted. The pot experiment indicated that the concentrations of HOAc-extractable Cd and Ni in soil reduced 6.26-15.33% and 13.31-19.53% with the inoculation of L5 and L6. In addition, the immobilization rates on Cd and Ni improved 61.27-128.50% and 23.69-39.66% with re-inoculation of strains L5 and L6 at 30 days, respectively. After inoculation of strains L5 and L6 for 60 days, the activities of FDA hydrolysis, acid phosphatase, urease, invertase and dehydrogenase in soil increased obviously. Furthermore, bacterial diversity indexes and community structure of soil were also improved. Thus, given the beneficial remediation effects of the isolated strains, L5 and L6 have great potentials for heavy metals contaminated soil remediation.


Assuntos
Bactérias/metabolismo , Cádmio/metabolismo , Níquel/metabolismo , Poluentes do Solo/metabolismo , Bactérias/enzimologia , Bactérias/isolamento & purificação , Cádmio/análise , Recuperação e Remediação Ambiental , Níquel/análise , Solo/química , Microbiologia do Solo , Poluentes do Solo/análise
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