Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.739
Filtrar
2.
Nat Commun ; 12(1): 3690, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140468

RESUMO

CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.


Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroidetes/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/genética , Bacteroidetes/enzimologia , Biologia Computacional , DNA Primase/genética , Primers do DNA/biossíntese , DNA Polimerase Dirigida por DNA/genética , Dimerização , Escherichia coli/metabolismo , Expressão Gênica , Mutação , Filogenia , Células Procarióticas/metabolismo , Proteínas Recombinantes , Ribonucleotídeos/metabolismo
3.
Nat Commun ; 12(1): 3867, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162839

RESUMO

Enzymes can evolve new catalytic activity when environmental changes present them with novel substrates. Despite this seemingly straightforward relationship, factors other than the direct catalytic target can also impact adaptation. Here, we characterize the catalytic activity of a recently evolved bacterial methyl-parathion hydrolase for all possible combinations of the five functionally relevant mutations under eight different laboratory conditions (in which an alternative divalent metal is supplemented). The resultant adaptive landscapes across this historical evolutionary transition vary in terms of both the number of "fitness peaks" as well as the genotype(s) at which they are found as a result of genotype-by-environment interactions and environment-dependent epistasis. This suggests that adaptive landscapes may be fluid and molecular adaptation is highly contingent not only on obvious factors (such as catalytic targets), but also on less obvious secondary environmental factors that can direct it towards distinct outcomes.


Assuntos
Adaptação Fisiológica/genética , Bactérias/genética , Proteínas de Bactérias/genética , Epistasia Genética , Hidrolases/genética , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Evolução Molecular , Interação Gene-Ambiente , Genótipo , Hidrolases/química , Hidrolases/metabolismo , Cinética , Metais/química , Metais/metabolismo , Metil Paration/química , Metil Paration/metabolismo , Mutação , Domínios Proteicos , Homologia de Sequência de Aminoácidos
4.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072989

RESUMO

Under anaerobic conditions, bacteria may utilize nitrates and nitrites as electron acceptors. Sensitivity to nitrous compounds is achieved via several mechanisms, some of which rely on sensor histidine kinases (HKs). The best studied nitrate- and nitrite-sensing HKs (NSHKs) are NarQ and NarX from Escherichia coli. Here, we review the function of NSHKs, analyze their natural diversity, and describe the available structural information. In particular, we show that around 6000 different NSHK sequences forming several distinct clusters may now be found in genomic databases, comprising mostly the genes from Beta- and Gammaproteobacteria as well as from Bacteroidetes and Chloroflexi, including those from anaerobic ammonia oxidation (annamox) communities. We show that the architecture of NSHKs is mostly conserved, although proteins from Bacteroidetes lack the HAMP and GAF-like domains yet sometimes have PAS. We reconcile the variation of NSHK sequences with atomistic models and pinpoint the structural elements important for signal transduction from the sensor domain to the catalytic module over the transmembrane and cytoplasmic regions spanning more than 200 Å.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias , Histidina Quinase , Proteínas de Membrana , Nitratos/metabolismo , Nitritos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Histidina Quinase/química , Histidina Quinase/classificação , Histidina Quinase/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Domínios Proteicos
5.
Nucleic Acids Res ; 49(11): 6027-6042, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-33905522

RESUMO

Type IIA topoisomerases catalyze a variety of different reactions: eukaryotic topoisomerase II relaxes DNA in an ATP-dependent reaction, whereas the bacterial representatives gyrase and topoisomerase IV (Topo IV) preferentially introduce negative supercoils into DNA (gyrase) or decatenate DNA (Topo IV). Gyrase and Topo IV perform separate, dedicated tasks during replication: gyrase removes positive supercoils in front, Topo IV removes pre-catenanes behind the replication fork. Despite their well-separated cellular functions, gyrase and Topo IV have an overlapping activity spectrum: gyrase is also able to catalyze DNA decatenation, although less efficiently than Topo IV. The balance between supercoiling and decatenation activities is different for gyrases from different organisms. Both enzymes consist of a conserved topoisomerase core and structurally divergent C-terminal domains (CTDs). Deletion of the entire CTD, mutation of a conserved motif and even by just a single point mutation within the CTD converts gyrase into a Topo IV-like enzyme, implicating the CTDs as the major determinant for function. Here, we summarize the structural and mechanistic features that make a type IIA topoisomerase a gyrase or a Topo IV, and discuss the implications for type IIA topoisomerase evolution.


Assuntos
DNA Girase/química , DNA Topoisomerase IV/química , Bactérias/enzimologia , DNA/química , DNA/metabolismo , DNA Girase/genética , DNA Girase/metabolismo , DNA Topoisomerase IV/genética , DNA Topoisomerase IV/metabolismo , DNA Topoisomerases Tipo II/química , Evolução Molecular , Conformação Proteica , Domínios Proteicos
6.
Molecules ; 26(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920799

RESUMO

With the relentless development of drug resistance and re-emergence of many pathogenic bacteria, the need for new antibiotics and new antibiotic targets is urgent and growing. Bacterial peptidyl-tRNA hydrolase, Pth1, is emerging as a promising new target for antibiotic development. From the conserved core and high degree of structural similarity, broad-spectrum inhibition is postulated. However, Pth1 small-molecule inhibition is still in the earliest stages. Focusing on pathogenic bacteria, herein we report the phylogenetic classification of Pth1 and natural product inhibition spanning phylogenetic space. While broad-spectrum inhibition is found, narrow-spectrum and even potentially clade-specific inhibition is more frequently observed. Additionally reported are enzyme kinetics and general in vitro Pth1 solubility that follow phylogenetic boundaries along with identification of key residues in the gate loop region that appear to govern both. The studies presented here demonstrate the sizeable potential for small-molecule inhibition of Pth1, improve understanding of Pth enzymes, and advance Pth1 as a much-needed novel antibiotic target.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Antibacterianos/farmacologia , Bactérias/enzimologia , Cinética , Filogenia , Solubilidade , Especificidade por Substrato
7.
Biochemistry (Mosc) ; 86(Suppl 1): S24-S37, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33827398

RESUMO

The review focuses on bacterial metallo-ß-lactamases (MßLs) responsible for the inactivation of ß-lactams and associated antibiotic resistance. The diversity of the active site structure in the members of different MßL subclasses explains different mechanisms of antibiotic hydrolysis and should be taken into account when searching for potential MßL inhibitors. The review describes the features of the antibiotic inactivation mechanisms by various MßLs studied by X-ray crystallography, NMR, kinetic measurements, and molecular modeling. The mechanisms of enzyme inhibition for each MßL subclass are discussed.


Assuntos
Domínio Catalítico , Resistência Microbiana a Medicamentos , beta-Lactamases/metabolismo , Antibacterianos/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrólise , Cinética , Modelos Moleculares , Conformação Proteica , beta-Lactamases/química
8.
J Food Sci ; 86(6): 2410-2420, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33904174

RESUMO

Tofu prepared by conventional methods often has a bitter taste and poor water-holding capacity (WHC). To improve the quality of the product, alternative processes must be developed. Herein, the effect of ultrasound pretreatment on the properties of soymilk and tofu gel derived thereof were investigated. Treatment of soymilk with ultrasound gave rise to a reduction in the particle size and an enhancement in the surface hydrophobicity, whereby optimum values were obtained after 15 min treatment. Subsequently, microbial transglutaminase (MTG) was added to ultrasound-treated soymilk to promote the soy protein crosslinking. The gel strength, WHC, and nonfreezable water content of MTG-catalyzed tofu gel obtained from treated soymilk increased with the extension of the ultrasound pretreatment time, whereas the free sulfhydryl content decreased because of the formation of disulfide bonds. Fourier transform infrared spectroscopy demonstrated variations in the secondary structure of MTG-catalyzed tofu gel. Furthermore, soymilk's exposure to high-intensity ultrasound pretreatment led to a tofu gel with a dense, homogenous, and stable network structure, as evidenced by scanning electron microscopy. Therefore, this study answers for the theoretical support of the industrial production of MTG-catalyzed tofu gel from ultrasound-treated soymilk. PRACTICAL APPLICATION: High-intensity ultrasound pretreatment improved the texture properties of MTG-catalyzed tofu gel. The resulting MTG-catalyzed tofu gel has potential application in industrial production.


Assuntos
Géis/química , Alimentos de Soja/análise , Leite de Soja/química , Proteínas de Soja/química , Transglutaminases/farmacologia , Ondas Ultrassônicas , Bactérias/enzimologia , Catálise , Géis/metabolismo , Géis/efeitos da radiação , Espectroscopia de Infravermelho com Transformada de Fourier
9.
Microbes Environ ; 36(2)2021.
Artigo em Inglês | MEDLINE | ID: mdl-33907062

RESUMO

Malodorous emissions are a crucial and inevitable issue during the decomposition of biological waste and contain a high concentration of ammonia. Biofiltration technology is a feasible, low-cost, energy-saving method that reduces and eliminates malodors without environmental impact. In the present study, we evaluated the effectiveness of compost from cattle manure and food waste as deodorizing media based on their removal of ammonia and the expression of ammonia-oxidizing genes, and identified the bacterial and archaeal communities in these media. Ammonia was removed by cattle manure compost, but not by food waste compost. The next-generation sequencing of 16S ribosomal RNA obtained from cattle manure compost revealed the presence of ammonia-oxidizing bacteria (AOB), including Cytophagia, Alphaproteobacteria, and Gammaproteobacteria, and ammonia-oxidizing archaea (AOA), such as Thaumarchaeota. In cattle manure compost, the bacterial and archaeal ammonia monooxygenase A (amoA) genes were both up-regulated after exposure to ammonia (fold ratio of 14.2±11.8 after/before), and the bacterial and archaeal communities were more homologous after than before exposure to ammonia, which indicates the adaptation of these communities to ammonia. These results suggest the potential of cattle manure compost as an efficient biological deodorization medium due to the activation of ammonia-oxidizing microbes, such as AOB and AOA, and the up-regulation of their amoA genes.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Esterco/microbiologia , Oxirredutases/metabolismo , Amônia/metabolismo , Animais , Archaea/classificação , Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/genética , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Bovinos , Compostagem , Filtração , Esterco/análise , Oxirredução , Oxirredutases/genética , Filogenia
10.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805379

RESUMO

ß-Glucosidase is a microbial cellulose multienzyme that plays an important role in the regulation of the entire cellulose hydrolysis process, which is the rate-limiting step in bacterial carbon cycling in marine environments. Despite its importance in coral reefs, the diversity of ß-glucosidase-producing bacteria, their genes, and enzymatic characteristics are poorly understood. In this study, 87 ß-glucosidase-producing cultivable bacteria were screened from 6 genera of corals. The isolates were assigned to 21 genera, distributed among three groups: Proteobacteria, Firmicutes, and Actinobacteria. In addition, metagenomics was used to explore the genetic diversity of bacterial ß-glucosidase enzymes associated with scleractinian corals, which revealed that these enzymes mainly belong to the glycosidase hydrolase family 3 (GH3). Finally, a novel recombinant ß-glucosidase, referred to as Mg9373, encompassing 670 amino acids and a molecular mass of 75.2 kDa, was classified as a member of the GH3 family and successfully expressed and characterized. Mg9373 exhibited excellent tolerance to ethanol, NaCl, and glucose. Collectively, these results suggest that the diversity of ß-glucosidase-producing bacteria and genes associated with scleractinian corals is high and novel, indicating great potential for applications in the food industry and agriculture.


Assuntos
Antozoários/microbiologia , Bactérias/enzimologia , Microbiota/genética , beta-Glucosidase/genética , Animais , Bactérias/genética , Metagenômica , Filogenia , beta-Glucosidase/metabolismo
11.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809335

RESUMO

The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in ß-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A ß-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on ß-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of ß-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.


Assuntos
Proteínas Mutantes/genética , Conformação Proteica , Engenharia de Proteínas , beta-Lactamases/genética , Aminoácidos/genética , Bactérias/enzimologia , Sítios de Ligação/genética , Domínio Catalítico/genética , Biologia Computacional , Estabilidade Enzimática/genética , Escherichia coli/enzimologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , beta-Lactamases/ultraestrutura
12.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799806

RESUMO

Carbonic anhydrases (CAs) have been identified as ideal catalysts for CO2 sequestration. Here, we report the sequence and structural analyses as well as the molecular dynamics (MD) simulations of four γ-CAs from thermophilic bacteria. Three of these, Persephonella marina, Persephonella hydrogeniphila, and Thermosulfidibacter takaii originate from hydrothermal vents and one, Thermus thermophilus HB8, from hot springs. Protein sequences were retrieved and aligned with previously characterized γ-CAs, revealing differences in the catalytic pocket residues. Further analysis of the structures following homology modeling revealed a hydrophobic patch in the catalytic pocket, presumed important for CO2 binding. Monitoring of proton shuttling residue His69 (P. marina γ-CA numbering) during MD simulations of P. hydrogeniphila and P. marina's γ-CAs (γ-PhCA and γ-PmCA), showed a different behavior to that observed in the γ-CA of Escherichia coli, which periodically coordinates Zn2+. This work also involved the search for hotspot residues that contribute to interface stability. Some of these residues were further identified as key in protein communication via betweenness centrality metric of dynamic residue network analysis. T. takaii's γ-CA showed marginally lower thermostability compared to the other three γ-CA proteins with an increase in conformations visited at high temperatures being observed. Hydrogen bond analysis revealed important interactions, some unique and others common in all γ-CAs, which contribute to interface formation and thermostability. The seemingly thermostable γ-CA from T. thermophilus strangely showed increased unsynchronized residue motions at 423 K. γ-PhCA and γ-PmCA were, however, preliminarily considered suitable as prospective thermostable CO2 sequestration agents.


Assuntos
Proteínas de Bactérias/metabolismo , Biomineralização , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dióxido de Carbono/química , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Catálise , Domínio Catalítico , Simulação por Computador , Fontes Termais/microbiologia , Fontes Hidrotermais/microbiologia , Simulação de Dinâmica Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Temperatura , Thermus thermophilus/enzimologia
13.
Microbes Environ ; 36(1)2021.
Artigo em Inglês | MEDLINE | ID: mdl-33716237

RESUMO

Bacterial extracellular aminopeptidases are key enzymes in protein processing in oligotrophic seawater. To the best of our knowledge, the regulation of aminopeptidase production in microbes inhabiting seawater has not yet been reported. The present study attempted to experimentally clarify which organic materials affect bacterial extracellular aminopeptidase production by nutrient-rich and starved cells growing in artificial seawater using Photobacterium, Alteromonas, Ruegeria, and Sulfitobacter. In all four species, we found that peptides induced bacterial extracellular aminopeptidase production. Amino acids led to cell growth with markedly lower aminopeptidase production by Photobacterium and Sulfitobacter, but not by Alteromonas and Ruegeria. These results suggest that the extracellular aminopeptidases of marine bacteria are primarily produced on demand in response to the presence of relevant substrates (peptides) in seawater. Peptidyl substances may be regulatory nutrients for marine bacterial growth in aquatic environments.


Assuntos
Aminopeptidases/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Espaço Extracelular/enzimologia , Peptídeos/metabolismo , Água do Mar/microbiologia , Aminopeptidases/genética , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Espaço Extracelular/genética , Filogenia
14.
J Enzyme Inhib Med Chem ; 36(1): 819-830, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33757387

RESUMO

Compounds containg catechol or bisphosphonate were tested as inhibitors of the zinc metalloproteases, thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) which are bacterial virulence factors, and the human matrix metalloproteases MMP-9 and -14. Inhibition of virulence is a putative strategy in the development of antibacterial drugs, but the inhibitors should not interfere with human enzymes. Docking indicated that the inhibitors bound MMP-9 and MMP-14 with the phenyl, biphenyl, chlorophenyl, nitrophenyl or methoxyphenyl ringsystem in the S1'-subpocket, while these ringsystems entered the S2'- or S1 -subpockets or a region involving amino acids in the S1'- and S2'-subpockets of the bacterial enzymes. An arginine conserved among the bacterial enzymes seemed to hinder entrance deeply into the S1'-subpocket. Only the bisphosphonate containing compound RC2 bound stronger to PLN and TLN than to MMP-9 and MMP-14. Docking indicated that the reason was that the conserved arginine (R203 in TLN and R198 in PLN) interacts with phosphate groups of RC2.


Assuntos
Antibacterianos/farmacologia , Catecóis/farmacologia , Difosfonatos/farmacologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Bactérias/enzimologia , Catecóis/síntese química , Catecóis/química , Difosfonatos/síntese química , Difosfonatos/química , Humanos , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/química , Metaloendopeptidases/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Células THP-1
15.
Mol Biol Rep ; 48(3): 2687-2701, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33650078

RESUMO

An integral approach to decoding both culturable and uncultured microorganisms' metabolic activity involves the whole genome sequencing (WGS) of individual/complex microbial communities. WGS of culturable microbes, amplicon sequencing, metagenomics, and single-cell genome analysis are selective techniques integrating genetic information and biochemical mechanisms. These approaches transform microbial biotechnology into a quick and high-throughput culture-independent evaluation and exploit pollutant-degrading microbes. They are windows into enzyme regulatory bioremediation pathways (i.e., dehalogenase) and the complete bioremediation process of organohalide pollutants. While the genome sequencing technique is gaining the scientific community's interest, it is still in its infancy in the field of pollutant bioremediation. The techniques are becoming increasingly helpful in unraveling and predicting the enzyme structure and explore metabolic and biodegradation capabilities.


Assuntos
Bactérias/enzimologia , Bactérias/genética , Hidrolases/biossíntese , Sequenciamento Completo do Genoma , Biodegradação Ambiental , Genoma Bacteriano , Hidrolases/química , Hidrolases/genética , Hidrolases/metabolismo , Metagenômica
16.
Arch Microbiol ; 203(5): 1953-1969, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33710379

RESUMO

One of the most abundant natural polymers on earth, chitin is a fibrous and structural polysaccharide, composed of N-acetyl-D-glucosamine. The biopolymer is the major structural constituent of fungi, arthropods, mollusks, nematodes, and some algae. The biodegradation of chitin is largely manifested by chitinolytic enzyme secreting organisms including bacteria, insects, and plants. Among them, bacterial chitinases represent the most promising, inexpensive, and sustainable source of proteins that can be employed for industrial-scale applications. To this end, the presented review comes at a timely moment to highlight the major sources of chitinolytic bacteria. It also discusses the potential pros and cons of prospecting bacterial chitinases that can be easily manipulated through genetic engineering. Additionally, we have elaborated the recent applications of the chitin thereby branding chitinases as potential candidates for biorefinery and biomedical research for eco-friendly and sustainable management of chitin waste in the environment.


Assuntos
Bactérias/metabolismo , Bioprospecção , Quitina/metabolismo , Quitinases/metabolismo , Acetilglucosamina/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Quitina/química , Quitinases/genética , Engenharia Genética
17.
Nat Commun ; 12(1): 1732, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741980

RESUMO

Macrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides' macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.


Assuntos
Antibacterianos/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Esterases/química , Esterases/genética , Macrolídeos/química , Antibacterianos/farmacologia , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Eritromicina/química , Genes Bacterianos , Macrolídeos/farmacologia , Simulação de Acoplamento Molecular , Conformação Proteica , Difração de Raios X
18.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669078

RESUMO

Gyrase is a bacterial type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme is essential in bacteria and is a validated drug target in the treatment of bacterial infections. Inhibition of gyrase activity is achieved by competitive inhibitors that interfere with ATP- or DNA-binding, or by gyrase poisons that stabilize cleavage complexes of gyrase covalently bound to the DNA, leading to double-strand breaks and cell death. Many of the current inhibitors suffer from severe side effects, while others rapidly lose their antibiotic activity due to resistance mutations, generating an unmet medical need for novel, improved gyrase inhibitors. DNA supercoiling by gyrase is associated with a series of nucleotide- and DNA-induced conformational changes, yet the full potential of interfering with these conformational changes as a strategy to identify novel, improved gyrase inhibitors has not been explored so far. This review highlights recent insights into the mechanism of DNA supercoiling by gyrase and illustrates the implications for the identification and development of conformation-sensitive and allosteric inhibitors.


Assuntos
DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Bactérias/enzimologia , Modelos Moleculares , Inibidores da Topoisomerase II/química
19.
Chem Commun (Camb) ; 57(20): 2441-2463, 2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33605953

RESUMO

Chorismate and isochorismate represent an important branching point connecting primary and secondary metabolism in bacteria, fungi, archaea and plants. Chorismate- and isochorismate-converting enzymes are potential targets for new bioactive compounds, as well as valuable biocatalysts for the in vivo and in vitro synthesis of fine chemicals. The diversity of the products of chorismate- and isochorismate-converting enzymes is reflected in the enzymatic three-dimensional structures and molecular mechanisms. Due to the high reactivity of chorismate and its derivatives, these enzymes have evolved to be accurately tailored to their respective reaction; at the same time, many of them exhibit a fascinating flexibility regarding side reactions and acceptance of alternative substrates. Here, we give an overview of the different (sub)families of chorismate- and isochorismate-converting enzymes, their molecular mechanisms, and three-dimensional structures. In addition, we highlight important results of mutagenetic approaches that generate a broader understanding of the influence of distinct active site residues for product formation and the conversion of one subfamily into another. Based on this, we discuss to what extent the recent advances in the field might influence the general mechanistic understanding of chorismate- and isochorismate-converting enzymes. Recent discoveries of new chorismate-derived products and pathways, as well as biocatalytic conversions of non-physiological substrates, highlight how this vast field is expected to continue developing in the future.


Assuntos
Ácido Corísmico/química , Ácido Corísmico/metabolismo , Transferases Intramoleculares/metabolismo , Oxo-Ácido-Liases/metabolismo , Bactérias/enzimologia , Bactérias/genética , Biocatálise , Domínio Catalítico , Cinética , Estrutura Molecular , Plantas/enzimologia , Plantas/genética , Ligação Proteica , Relação Estrutura-Atividade
20.
Microbiome ; 9(1): 43, 2021 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-33583433

RESUMO

BACKGROUND: Chitin ranks as the most abundant polysaccharide in the oceans yet knowledge of shifts in structure and diversity of chitin-degrading communities across marine niches is scarce. Here, we integrate cultivation-dependent and -independent approaches to shed light on the chitin processing potential within the microbiomes of marine sponges, octocorals, sediments, and seawater. RESULTS: We found that cultivatable host-associated bacteria in the genera Aquimarina, Enterovibrio, Microbulbifer, Pseudoalteromonas, Shewanella, and Vibrio were able to degrade colloidal chitin in vitro. Congruent with enzymatic activity bioassays, genome-wide inspection of cultivated symbionts revealed that Vibrio and Aquimarina species, particularly, possess several endo- and exo-chitinase-encoding genes underlying their ability to cleave the large chitin polymer into oligomers and dimers. Conversely, Alphaproteobacteria species were found to specialize in the utilization of the chitin monomer N-acetylglucosamine more often. Phylogenetic assessments uncovered a high degree of within-genome diversification of multiple, full-length endo-chitinase genes for Aquimarina and Vibrio strains, suggestive of a versatile chitin catabolism aptitude. We then analyzed the abundance distributions of chitin metabolism-related genes across 30 Illumina-sequenced microbial metagenomes and found that the endosymbiotic consortium of Spongia officinalis is enriched in polysaccharide deacetylases, suggesting the ability of the marine sponge microbiome to convert chitin into its deacetylated-and biotechnologically versatile-form chitosan. Instead, the abundance of endo-chitinase and chitin-binding protein-encoding genes in healthy octocorals leveled up with those from the surrounding environment but was found to be depleted in necrotic octocoral tissue. Using cultivation-independent, taxonomic assignments of endo-chitinase encoding genes, we unveiled previously unsuspected richness and divergent structures of chitinolytic communities across host-associated and free-living biotopes, revealing putative roles for uncultivated Gammaproteobacteria and Chloroflexi symbionts in chitin processing within sessile marine invertebrates. CONCLUSIONS: Our findings suggest that differential chitin degradation pathways, utilization, and turnover dictate the processing of chitin across marine micro-niches and support the hypothesis that inter-species cross-feeding could facilitate the co-existence of chitin utilizers within marine invertebrate microbiomes. We further identified chitin metabolism functions which may serve as indicators of microbiome integrity/dysbiosis in corals and reveal putative novel chitinolytic enzymes in the genus Aquimarina that may find applications in the blue biotechnology sector. Video abstract.


Assuntos
Organismos Aquáticos/microbiologia , Bactérias/metabolismo , Quitina/metabolismo , Sedimentos Geológicos/microbiologia , Metagenômica , Microbiota , Água do Mar/microbiologia , Animais , Antozoários/microbiologia , Bactérias/enzimologia , Bactérias/genética , Quitinases/genética , Quitinases/metabolismo , Microbiota/genética , Oceanos e Mares , Filogenia , Poríferos/microbiologia , Simbiose
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...