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1.
PLoS One ; 15(8): e0226235, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797046

RESUMO

Plant-derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z. mobilis, we engineered a strain (Z. mobilis GH3) that expresses a glycosyl hydrolase (GH) with ß-glucosidase activity from a related α-proteobacterial species, Caulobacter crescentus, and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and extracellular release of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 h and use it for growth and ethanol production. Alternatively, pre-growth of Z. mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid release or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z. mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z. mobilis to sucrose that promotes sucrase production.


Assuntos
Celobiose/metabolismo , Zymomonas/crescimento & desenvolvimento , Zymomonas/metabolismo , Adaptação Fisiológica/fisiologia , Biomassa , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Celulose/metabolismo , Expressão Gênica/genética , Glucose/metabolismo , Hidrolases/metabolismo , Proteômica , Sacarase/metabolismo , Sacarose/metabolismo , Zymomonas/genética , beta-Glucosidase/metabolismo
2.
Nature ; 584(7821): 479-483, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788728

RESUMO

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Assuntos
Membrana Celular/química , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Virulência
3.
PLoS One ; 15(7): e0235785, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645074

RESUMO

The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.


Assuntos
Baratas/microbiologia , Conidiobolus/fisiologia , Ácidos Graxos/metabolismo , Proteínas Fúngicas/metabolismo , Hidrolases/metabolismo , Animais , Baratas/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Masculino
4.
Nat Commun ; 11(1): 2816, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499527

RESUMO

The intense arms race between bacteria and phages has led to the development of diverse antiphage defense systems in bacteria. Unlike well-known restriction-modification and CRISPR-Cas systems, recently discovered systems are poorly characterized. One such system is the Thoeris defense system, which consists of two genes, thsA and thsB. Here, we report structural and functional analyses of ThsA and ThsB. ThsA exhibits robust NAD+ cleavage activity and a two-domain architecture containing sirtuin-like and SLOG-like domains. Mutation analysis suggests that NAD+ cleavage is linked to the antiphage function of Thoeris. ThsB exhibits a structural resemblance to TIR domain proteins such as nucleotide hydrolases and Toll-like receptors, but no enzymatic activity is detected in our in vitro assays. These results further our understanding of the molecular mechanism underlying the Thoeris defense system, highlighting a unique strategy for bacterial antiphage resistance via NAD+ degradation.


Assuntos
Bacteriófagos/genética , Escherichia coli/virologia , NAD/metabolismo , Bacillus cereus/metabolismo , Sistemas CRISPR-Cas , Clonagem Molecular , Cristalografia por Raios X , Análise Mutacional de DNA , Escherichia coli/metabolismo , Hidrolases/metabolismo , Cinética , Mutação , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores Toll-Like/metabolismo
5.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 868-878, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567270

RESUMO

2-Haloacid dehalogenases (EC 3.8.1.X) catalyze the hydrolytic dehalogenation of 2-haloacids, releasing halogen ions and producing corresponding 2-hydroxyacids. The enzymes not only degrade xenobiotic halogenated pollutants, but also show wide substrate profile and astonishing efficiency for enantiomer resolution, making them valuable in environmental protection and the green synthesis of optically pure chiral compounds. A variety of 2-haloacid dehalogenases have been biochemically characterized so far. Further studies have been made in protein crystal structures and catalytic mechanisms. Here, we review the recent progresses of 2-haloacid dehalogenases in their source, protein structures, reaction mechanisms, catalytic properties and application. We also suggest further research directions for 2-haloacid dehalogenase.


Assuntos
Hidrolases , Catálise , Halogenação , Hidrolases/química , Hidrolases/metabolismo , Hidrólise , Pesquisa/tendências , Especificidade por Substrato
6.
Chem Biol Interact ; 327: 109181, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32569593

RESUMO

Acute pancreatitis (AP) is a sudden pancreatic inflammation accompanied by an excessive reactive oxygen species production that provokes inflammation. The present study investigated whether carvedilol can protect against l-arginine induced AP in a rat model and studied the mechanisms associated with its protection. Rats were divided into four groups: a control group, an AP group (injected with 2 doses of l-arginine 250 mg/100 g body weight at 1 h interval, intraperitoneally) on the 22nd day of the experiment, a carvedilol group (10 mg/kg, orally) for 21 successive days, and finally a carvedilol + AP group. It was found that pretreatment with carvedilol decreased α-amylase and lipase activities as well as C-reactive protein (CRP) and malondialdehyde levels; on the other hand, it improved the reduced glutathione (GSH) level and catalase (CAT) activity. In addition, carvedilol markedly decreased all of the following biomarkers: nuclear factor kappa B (NF-κB p65), p38 mitogen-activated protein kinases (P38-MAPK), signal transducer and activator of transcription 1 (STAT1-α), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), myeloperoxidase (MPO), and phospholipase A2 (PLA2) levels that was induced by l-arginine. Finally, carvedilol noticeably down regulated the pancreatitis associated protein (PAP2) and the pancreas platelets activating factor (PAF) genes expression. In conclusion: carvedilol protected against l-arginine induced AP in rats, via the inhibition of cellular oxidative stress and inflammatory pathways that contributed to pancreas injury.


Assuntos
Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Carvedilol/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Pancreatite/tratamento farmacológico , Animais , Arginina , Biomarcadores/metabolismo , Proteína C-Reativa/metabolismo , Glutationa/metabolismo , Hidrolases/metabolismo , Interleucina-1beta/metabolismo , Masculino , Malondialdeído/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/metabolismo , Peroxidase/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismo
7.
Nature ; 580(7802): 216-219, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269349

RESUMO

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.


Assuntos
Hidrolases/química , Hidrolases/metabolismo , Plásticos/química , Plásticos/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Engenharia de Proteínas , Reciclagem , Actinobacteria/enzimologia , Burkholderiales/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Ensaios Enzimáticos , Estabilidade Enzimática , Fusarium/enzimologia , Modelos Moleculares , Ácidos Ftálicos/metabolismo , Polimerização
8.
PLoS Pathog ; 16(4): e1007979, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32298382

RESUMO

Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/crescimento & desenvolvimento , Brucelose/metabolismo , Hidrolases/metabolismo , NAD/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de Virulência/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Brucella abortus/metabolismo , Brucelose/microbiologia , Células HeLa , Humanos , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Virulência/genética
9.
Nat Commun ; 11(1): 1867, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313070

RESUMO

Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)- and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (-)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (-)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.


Assuntos
Alcaloides/biossíntese , Compostos Ferrosos/metabolismo , Hidrolases/metabolismo , Ácidos Cetoglutáricos/metabolismo , Menispermaceae/metabolismo , Compostos de Espiro/metabolismo , Alcaloides/química , Alcaloides/genética , Bactérias/metabolismo , Biocatálise , Genes de Plantas/genética , Halogenação , Menispermaceae/embriologia , Menispermaceae/genética , Mutagênese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes , Metabolismo Secundário/genética , Alinhamento de Sequência , Compostos de Espiro/química , Transcriptoma
10.
Nat Commun ; 11(1): 1264, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152287

RESUMO

Wildfires can encourage the establishment of invasive plants by releasing potent germination stimulants, such as karrikins. Seed germination of Brassica tournefortii, a noxious weed of Mediterranean climates, is strongly stimulated by KAR1, the archetypal karrikin produced from burning vegetation. In contrast, the closely-related yet non-fire-associated ephemeral Arabidopsis thaliana is unusual because it responds preferentially to KAR2. The α/ß-hydrolase KARRIKIN INSENSITIVE 2 (KAI2) is the putative karrikin receptor identified in Arabidopsis. Here we show that B. tournefortii expresses three KAI2 homologues, and the most highly-expressed homologue is sufficient to confer enhanced responses to KAR1 relative to KAR2 when expressed in Arabidopsis. We identify two amino acid residues near the KAI2 active site that explain the ligand selectivity, and show that this combination has arisen independently multiple times within dicots. Our results suggest that duplication and diversification of KAI2 proteins could confer differential responses to chemical cues produced by environmental disturbance, including fire.


Assuntos
Arabidopsis/metabolismo , Brassica/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Furanos/farmacologia , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/metabolismo , Piranos/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis , Brassica/efeitos dos fármacos , Brassica/genética , Domínio Catalítico , Fogo , Regulação da Expressão Gênica de Plantas , Germinação/efeitos dos fármacos , Germinação/fisiologia , Hidrolases/genética , Hidrolases/metabolismo , Magnoliopsida , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plântula , Sementes/efeitos dos fármacos , Sementes/fisiologia , Análise de Sequência de Proteína , Transcriptoma , Incêndios Florestais
11.
Nat Commun ; 11(1): 1468, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193369

RESUMO

The α-glucosidase inhibitor acarbose, produced by Actinoplanes sp. SE50/110, is a well-known drug for the treatment of type 2 diabetes mellitus. However, the largely unexplored biosynthetic mechanism of this compound has impeded further titer improvement. Herein, we uncover that 1-epi-valienol and valienol, accumulated in the fermentation broth at a strikingly high molar ratio to acarbose, are shunt products that are not directly involved in acarbose biosynthesis. Additionally, we find that inefficient biosynthesis of the amino-deoxyhexose moiety plays a role in the formation of these shunt products. Therefore, strategies to minimize the flux to the shunt products and to maximize the supply of the amino-deoxyhexose moiety are implemented, which increase the acarbose titer by 1.2-fold to 7.4 g L-1. This work provides insights into the biosynthesis of the C7-cyclitol moiety and highlights the importance of assessing shunt product accumulation when seeking to improve the titer of microbial pharmaceutical products.


Assuntos
Acarbose/metabolismo , Vias Biossintéticas , Actinomycetales/metabolismo , Biocatálise , Vias Biossintéticas/genética , Ciclitóis , Fermentação , Hexoses , Hidrolases/metabolismo , Engenharia Metabólica , Análise do Fluxo Metabólico , Família Multigênica , Fosforilação
12.
Genes Dev ; 34(5-6): 263-284, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32029451

RESUMO

ADP-ribosylation is an intricate and versatile posttranslational modification involved in the regulation of a vast variety of cellular processes in all kingdoms of life. Its complexity derives from the varied range of different chemical linkages, including to several amino acid side chains as well as nucleic acids termini and bases, it can adopt. In this review, we provide an overview of the different families of (ADP-ribosyl)hydrolases. We discuss their molecular functions, physiological roles, and influence on human health and disease. Together, the accumulated data support the increasingly compelling view that (ADP-ribosyl)hydrolases are a vital element within ADP-ribosyl signaling pathways and they hold the potential for novel therapeutic approaches as well as a deeper understanding of ADP-ribosylation as a whole.


Assuntos
ADP-Ribosilação/fisiologia , Difosfato de Adenosina/metabolismo , Hidrolases/química , Hidrolases/metabolismo , Humanos , Hidrolases/classificação , Domínios Proteicos , Relação Estrutura-Atividade
13.
PLoS One ; 15(2): e0229376, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084230

RESUMO

The alpha/beta-Hydrolases (ABH) are a structural class of proteins that are found widespread in nature and includes enzymes that can catalyze various reactions in different substrates. The catalytic versatility of the ABH fold enzymes, which has been a valuable property in protein engineering applications, is based on a similar acid-base-nucleophile catalytic mechanism. In our research, we are concerned with the structure that surrounds the key units of the catalytic machinery, and we have previously found conserved structural organizations that coordinate the catalytic acid, the catalytic nucleophile and the residues of the oxyanion hole. Here, we explore the architecture that surrounds the catalytic histidine at the active sites of enzymes from 40 ABH fold families, where we have identified six conserved interactions that coordinate the catalytic histidine next to the catalytic acid and the catalytic nucleophile. Specifically, the catalytic nucleophile is coordinated next to the catalytic histidine by two weak hydrogen bonds, while the catalytic acid is directly involved in the coordination of the catalytic histidine through by two weak hydrogen bonds. The imidazole ring of the catalytic histidine is coordinated by a CH-π contact and a hydrophobic interaction. Moreover, the catalytic triad residues are connected with a residue that is located at the core of the active site of ABH fold, which is suggested to be the fourth member of a "structural catalytic tetrad". Besides their role in the stability of the catalytic mechanism, the conserved elements of the catalytic site are actively involved in ligand binding and affect other properties of the catalytic activity, such as substrate specificity, enantioselectivity, pH optimum and thermostability of ABH fold enzymes. These properties are regularly targeted in protein engineering applications, and thus, the identified conserved structural elements can serve as potential modification sites in order to develop ABH fold enzymes with altered activities.


Assuntos
Histidina/química , Hidrolases/química , Modelos Moleculares , Sítios de Ligação , Catálise , Domínio Catalítico , Histidina/metabolismo , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Hidrolases/metabolismo , Especificidade por Substrato
14.
PLoS One ; 15(2): e0229141, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084175

RESUMO

The objective was to evaluate the effect of the addition of 0, 10, 20, and 30 mg.kg-1 of natural matter of a lignocellulosic enzymatic complex produced by the white rot fungus on the chemical composition, cumulative gas production in vitro, and antioxidant compounds of corn and sugarcane silages. After being chopped and treated with the enzymatic complex, the plants were packed in vacuum-sealed bags. After 60 days, the mini silos were opened and the samples were dried in a forced ventilation oven at 55 °C for analysis of the proposed parameters. The experiment was conducted in a completely randomized design with four replicates per treatment. In the corn silage, there was a linear reduction in the lignin concentration. In the sugarcane silage showed a reduction of 12% in the lignin concentration, a linear reduction in the hemicellulose content, and a decrease of 8% in the cellulose concentration compared to the control treatment. The lignin monomers had linear increases in the syringyl:guaiacil ratio. This reflected on significant increases in the concentration of the non-fibrous carbohydrates and the A + B1 fraction of the carbohydrates, and a reduction in the C fraction. The in vitro gas production increased, the time of colonization and initiation of in vitro fermentation linearly decreased in both silages. The phenolic compounds and the antioxidant capacity increased linearly with the addition of the enzymes in both silages. The addition of the lignocellulolytic enzymes to the silages caused changes in the cell wall, resulting in improvements in the in vitro fermentative parameters, besides the additional effect on the antioxidant capacity. There was an effect of the addition of the enzymes on the evaluated fodder, and the best concentration was, on average, 20 mg kg-1 MN for corn silage and 10 mg kg-1 NM for sugarcane silage.


Assuntos
Antioxidantes/análise , Hidrolases/metabolismo , Lignina/análise , Pleurotus/metabolismo , Saccharum/metabolismo , Silagem/análise , Zea mays/metabolismo , Hidrolases/biossíntese , Hidrólise , Lignina/metabolismo
15.
Appl Environ Microbiol ; 86(9)2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32086305

RESUMO

The quinolone ring is a common core structure of natural products exhibiting antimicrobial, cytotoxic, and signaling activities. A prominent example is the Pseudomonas quinolone signal (PQS), a quorum-sensing signal molecule involved in the regulation of virulence of Pseudomonas aeruginosa The key reaction to quinolone inactivation and biodegradation is the cleavage of the 3-hydroxy-4(1H)-quinolone ring, catalyzed by dioxygenases (HQDs), which are members of the α/ß-hydrolase fold superfamily. The α/ß-hydrolase fold core domain consists of a ß-sheet surrounded by α-helices, with an active site usually containing a catalytic triad comprising a nucleophilic residue, an acidic residue, and a histidine. The nucleophile is located at the tip of a sharp turn, called the "nucleophilic elbow." In this work, we developed a search workflow for the identification of HQD proteins from databases. Search and validation criteria include an [H-x(2)-W] motif at the nucleophilic elbow, an [HFP-x(4)-P] motif comprising the catalytic histidine, the presence of a helical cap domain, the positioning of the triad's acidic residue at the end of ß-strand 6, and a set of conserved hydrophobic residues contributing to the substrate cavity. The 161 candidate proteins identified from the UniProtKB database originate from environmental and plant-associated microorganisms from all domains of life. Verification and characterization of HQD activity of 9 new candidate proteins confirmed the reliability of the search strategy and suggested residues correlating with distinct substrate preferences. Among the new HQDs, PQS dioxygenases from Nocardia farcinica, N. cyriacigeorgica, and Streptomyces bingchenggensis likely are part of a catabolic pathway for alkylquinolone utilization.IMPORTANCE Functional annotation of protein sequences is a major requirement for the investigation of metabolic pathways and the identification of sought-after biocatalysts. To identify heterocyclic ring-cleaving dioxygenases within the huge superfamily of α/ß-hydrolase fold proteins, we defined search and validation criteria for the primarily motif-based identification of 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQD). HQDs are key enzymes for the inactivation of metabolites, which can have signaling, antimicrobial, or cytotoxic functions. The HQD candidates detected in this study occur particularly in environmental and plant-associated microorganisms. Because HQDs active toward the Pseudomonas quinolone signal (PQS) likely contribute to interactions within microbial communities and modulate the virulence of Pseudomonas aeruginosa, we analyzed the catalytic properties of a PQS-cleaving subset of HQDs and specified characteristics to identify PQS-cleaving dioxygenases within the HQD family.


Assuntos
Proteínas de Bactérias/genética , Hidrolases/genética , Pseudomonas aeruginosa/genética , Quinolonas/metabolismo , Percepção de Quorum , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrolases/química , Hidrolases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Alinhamento de Sequência
16.
mBio ; 11(1)2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047134

RESUMO

Macrodomain (MD), a highly conserved protein fold present in a subset of plus-strand RNA viruses, binds to and hydrolyzes ADP-ribose (ADPr) from ADP-ribosylated proteins. ADPr-binding by the alphavirus nonstructural protein 3 (nsP3) MD is necessary for the initiation of virus replication in neural cells, whereas hydrolase activity facilitates replication complex amplification. To determine the importance of these activities for pathogenesis of alphavirus encephalomyelitis, mutations were introduced into the nsP3 MD of Sindbis virus (SINV), and the effects on ADPr binding and hydrolase activities, virus replication, immune responses, and disease were assessed. Elimination of ADPr-binding and hydrolase activities (G32E) severely impaired in vitro replication of SINV in neural cells and in vivo replication in the central nervous systems of 2-week-old mice with reversion to wild type (WT) (G) or selection of a less compromising change (S) during replication. SINVs with decreased binding and hydrolase activities (G32S and G32A) or with hydrolase deficiency combined with better ADPr-binding (Y114A) were less virulent than WT virus. Compared to the WT, the G32S virus replicated less well in both the brain and spinal cord, induced similar innate responses, and caused less severe disease with full recovery of survivors, whereas the Y114A virus replicated well, induced higher expression of interferon-stimulated and NF-κB-induced genes, and was cleared more slowly from the spinal cord with persistent paralysis in survivors. Therefore, MD function was important for neural cell replication both in vitro and in vivo and determined the outcome from alphavirus encephalomyelitis in mice.IMPORTANCE Viral encephalomyelitis is an important cause of long-term disability, as well as acute fatal disease. Identifying viral determinants of outcome helps in assessing disease severity and developing new treatments. Mosquito-borne alphaviruses infect neurons and cause fatal disease in mice. The highly conserved macrodomain of nonstructural protein 3 binds and can remove ADP-ribose (ADPr) from ADP-ribosylated proteins. To determine the importance of these functions for virulence, recombinant mutant viruses were produced. If macrodomain mutations eliminated ADPr-binding or hydrolase activity, viruses did not grow. If the binding and hydrolase activities were impaired, the viruses grew less well than the wild-type virus, induced similar innate responses, and caused less severe disease, and most of the infected mice recovered. If binding was improved, but hydrolase activity was decreased, the virus replicated well and induced greater innate responses than did the WT, but clearance from the nervous system was impaired, and mice remained paralyzed. Therefore, macrodomain function determined the outcome of alphavirus encephalomyelitis.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Hidrolases/metabolismo , Vírus Sindbis/genética , Vírus Sindbis/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Adenosina Difosfato Ribose/genética , Animais , Encéfalo/virologia , Linhagem Celular , Encefalomielite/virologia , Feminino , Hidrolases/genética , Camundongos , Mutação , Neurônios/virologia , Transdução de Sinais , Proteínas não Estruturais Virais/genética , Virulência , Replicação Viral/genética
17.
Enzyme Microb Technol ; 134: 109484, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044031

RESUMO

Xylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear ß-1,3-ß-1,4-xylan) from which also an isomeric xylotriose Xyl-ß-1,3-Xyl-ß-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.


Assuntos
Acremonium/enzimologia , Dissacarídeos/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Hidrolases/metabolismo , Acremonium/genética , Endo-1,4-beta-Xilanases/genética , Hidrolases/genética , Especificidade por Substrato
18.
Artigo em Inglês | MEDLINE | ID: mdl-31990641

RESUMO

Condensed phosphates are deliberately added to the fishery and processed marine food products on purpose to increase the weight of products. However, excessive intake overwhelming bodily homoeostasis can result in phosphate toxicity such as mineral and bone disorders, associated with chronic kidney diseases, and cardiovascular events. Rapid analysis for condensed phosphates is required to detect the illegal adulteration of processed marine products. We optimised an analytical method using ion chromatography for the rapid and selective detection of condensed phosphates in various kinds of fishery and processed marine products. We compared the performance of three columns (IonPac AS11, AS11-HC, and AS16) for the detection of condensed phosphates with respect to time of analysis and sensitivity. The IonPac AS11 column exhibited the shortest retention time for the major condensed phosphates (pyro-, tri-, and trimetaphosphate), the highest sensitivity for trimetaphosphate, and good repeatability and precision. Microwave and boiling processing were examined as methods to prevent hydrolysis of condensed phosphates into orthophosphate, which is critical in distinguishing intentionally added condensed phosphates from naturally occurring orthophosphate. Microwave treatment was determined to be the more effective method to suppress hydrolysis of condensed phosphates to orthophosphate. Furthermore, microwave processing could be used for thawing the frozen samples, saving extra effort and time. We confirmed that the method exhibits good recovery (80% or more) and precision (%RSD < 10%) for samples with various matrices. With the method, 14 kinds of fishery and processed marine food products were successfully analysed for condensed phosphates. Especially, we identified that phosphate levels in the processed shrimp and dried shredded squid samples exceeded the maximum allowable levels specified in the CODEX standard. We believe that our method would be useful for the rapid analysis of condensed phosphates in various types of fishery and processed marine food products.


Assuntos
Fosfatos/análise , Animais , Cromatografia por Troca Iônica , Pesqueiros , Hidrolases/metabolismo , Limite de Detecção , Micro-Ondas , Alimentos Marinhos , Sensibilidade e Especificidade , Extração em Fase Sólida
19.
Appl Microbiol Biotechnol ; 104(5): 2067-2077, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932896

RESUMO

Halohydrin dehalogenases (HHDHs) have attracted much attention due to their ability to synthesize enantiomerically enriched epoxides and ß-haloalcohols. However, most of the HHDHs exhibit low enantioselectivity. Here, a HHDH from the alphaproteobacteria isolate 46_93_T64 (AbHHDH), which shows only poor enantioselectivity in the catalytic resolution of rac-PGE (E = 9.9), has been subjected to protein engineering to enhance its enantioselectivity. Eight mutants (R89K, R89Y, V137I, P178A, N179Q, N179L, F187L, F187A) showed better enantioselectivity than the wild type. The best single mutant N179L (E = 93.0) showed a remarkable 9.4-fold increase in the enantioselectivity. Then, the single mutations were combined to produce the double, triple, quadruple, and quintuple mutants. Among the combinational mutants, the best variant (R89Y/N179L) showed an increased E value of up to 48. The E values of the variants N179L and R89Y/N179L for other epoxides 2-7 were 12.2 to > 200, which showed great improvement compared to 1.2 to 10.5 for the wild type. Using the variant N179L, enantiopure (R)-PGE with > 99% ee could be readily prepared, affording a high yield and a high concentration.


Assuntos
Proteínas de Bactérias/metabolismo , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Hidrolases/metabolismo , Alphaproteobacteria/enzimologia , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Hidrolases/química , Hidrolases/genética , Hidrolases/isolamento & purificação , Cinética , Modelos Moleculares , Mutação , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato
20.
Ecotoxicol Environ Saf ; 190: 110148, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911388

RESUMO

Phthalate esters have raised public concerns owing to their effects on the environment and human health. We identified a novel phthalate-degrading hydrolase, EstJ6, from a metagenomic library using function-driven screening. Phylogenetic analysis indicated that EstJ6 is a member of family IV esterases. EstJ6 hydrolyzed various dialkyl and monoalkyl phthalate esters, and exhibited high hydrolytic activity (128 U/mg) toward dibutyl phthalate at 40 °C and pH 7.5. EstJ6 hydrolyzed not only common phthalate esters with simple side chains but also diethylhexyl phthalate and monoethylhexyl phthalate, which have complex and long side chains. Site-directed mutagenesis indicated that the catalytic triad residues of EstJ6 consists of Ser146, Glu240, and His270. EstJ6 is therefore a promising biodegradation enzyme, and our study illustrates the advantages of a metagenomic approach in identifying enzyme-coding genes for agricultural, food, and biotechnological applications.


Assuntos
Biodegradação Ambiental , Hidrolases/metabolismo , Ácidos Ftálicos/metabolismo , Dibutilftalato/metabolismo , Dietilexilftalato/metabolismo , Esterases/metabolismo , Ésteres/química , Biblioteca Gênica , Hidrolases/genética , Hidrólise , Metagenoma , Filogenia , Solo
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