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1.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076933

RESUMO

Epidemiologic studies have shown associations between periodontitis and rheumatoid arthritis (RA), but a causal relationship has not been established. Citrullination of gingival proteins by human peptidylarginine deiminases (PADs) or PAD from Porphyromonas gingivalis has been proposed to generate autoantigens in anti-CCP-positive RA. This study investigated whether the association between periodontitis and RA is influenced by single nucleotide polymorphisms (SNPs) in the genes encoding PAD2 and PAD4 that catalyze aberrant citrullination in RA and often are overexpressed in inflamed gingival connective tissue in subjects with periodontitis. The study included 137 RA patients and 161 controls with self-reported periodontitis. Periodontitis onset preceded RA onset by 13 years on average and was not associated with any of the SNPs investigated. In subjects with periodontitis, carriage of the minor alleles of rs2057094 and rs2235912 in PADI2 significantly increased the risk of RA (odds ratios 1.42 [p = 0.03] and 1.48 [p = 0.02], respectively), and this effect was driven by the anti-CCP-negative RA patients. The minor alleles of these SNPs only increased risk of anti-CCP-positive RA in individuals with periodontitis and a history of smoking. These data suggest that individuals with periodontitis carrying the minor alleles of SNPs rs2057094, rs2076616 and rs2235912 in PADI2 may be at increased risk of RA.


Assuntos
Artrite Reumatoide , Periodontite , Anticorpos Anti-Proteína Citrulinada , Artrite Reumatoide/complicações , Artrite Reumatoide/genética , Autoanticorpos , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Periodontite/complicações , Periodontite/genética , Polimorfismo de Nucleotídeo Único , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo
2.
Sci Rep ; 12(1): 15502, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36109598

RESUMO

Gulosibacter molinativorax ON4T is the only known organism to produce molinate hydrolase (MolA), which catalyses the breakdown of the thiocarbamate herbicide into azepane-1-carboxylic acid (ACA) and ethanethiol. A combined genomic and transcriptomic strategy was used to fully characterize the strain ON4T genome, particularly the molA genetic environment, to identify the potential genes encoding ACA degradation enzymes. Genomic data revealed that molA is the only catabolic gene of a novel composite transposon (Tn6311), located in a novel low copy number plasmid (pARLON1) harbouring a putative T4SS of the class FATA. pARLON1 had an ANI value of 88.2% with contig 18 from Agrococcus casei LMG 22410T draft genome. Such results suggest that pARLON1 is related to genomic elements of other Actinobacteria, although Tn6311 was observed only in strain ON4T. Furthermore, genomic and transcriptomic data demonstrated that the genes involved in ACA degradation are chromosomal. Based on their overexpression when growing in the presence of molinate, the enzymes potentially involved in the heterocyclic ring breakdown were predicted. Among these, the activity of a protein related to caprolactone hydrolase was demonstrated using heterologous expression. However, further studies are needed to confirm the role of the other putative enzymes.


Assuntos
Actinomycetales , Herbicidas , Actinobacteria , Actinomycetales/genética , Azepinas , Herbicidas/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Tiocarbamatos
3.
Nat Commun ; 13(1): 5012, 2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-36008405

RESUMO

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.


Assuntos
Lesões Pré-Cancerosas , Tirosinemias , Animais , Modelos Animais de Doenças , Terapia Genética , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Cirrose Hepática/terapia , Nitrobenzoatos/farmacologia , Nitrobenzoatos/uso terapêutico , Suínos , Tirosinemias/genética , Tirosinemias/terapia
4.
Commun Biol ; 5(1): 816, 2022 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-35963893

RESUMO

TAR DNA-Binding Protein 43 (TDP-43) has been well studied in neurodegenerative diseases, but its potential role in malignance is still unclear. Here, we demonstrate that TDP-43 contributes to the suppression of apoptosis by facilitating lipid metabolism in hepatocellular carcinoma (HCC). In HCC cells, TDP-43 is able to suppress apoptosis while deletion of it markedly induces apoptosis. RNA-sequencing identifies the lipid metabolism gene abhydrolase domain containing 2 (ABHD2) as the target gene of TDP-43. Tissue microarray analysis shows the positive correlation of TDP-43 and ABHD2 in HCC. Mechanistically, TDP-43 binds with the UG-rich sequence1 of ABHD2 3'UTR to enhance the mRNA stability of ABHD2, thereby upregulating ABHD2. Afterwards, TDP-43 promotes the production of free fatty acid and fatty acid oxidation-originated reactive oxygen species (ROS) in an ABHD2-dependent manner, so as to suppress apoptosis of HCC. Our findings provide insights into the mechanism of HCC progression and reveal TDP-43/ABHD2 as potential targets for the precise treatment of HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Apoptose , Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Hidrolases/metabolismo , Metabolismo dos Lipídeos , Neoplasias Hepáticas/patologia
5.
BMC Microbiol ; 22(1): 190, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35922769

RESUMO

Recent studies have demonstrated the potential of surface display technology in therapeutic development and enzyme immobilization. Utilization of lactic acid bacteria in non-GMO surface display applications is advantageous due to its GRAS status. This study aimed to develop a novel, non-GMO cell wall anchoring system for lactic acid bacteria using a cell-surface hydrolase (CshA) from Lactiplantibacillus plantarum SK156 for potential industrial and biomedical applications. Analysis of the CshA revealed that it does not contain any known classical anchor domains. Although CshA lacks a classical anchor domain, it successfully displayed the reporter protein superfolder GFP on the surface of several lactic acid bacteria in host dependent manner. CshA-sfGFP fusion protein was displayed greatest on Limosilactobacillus fermentum SK152. Pretreatment with trichloroacetic acid further enhanced the binding of CshA to Lm. fermentum. The binding conditions of CshA on pretreated Lm. fermentum (NaCl, pH, time, and temperature) were also optimized, resulting in a maximum binding of up to 106 CshA molecules per pretreated Lm. fermentum cell. Finally, this study demonstrated that CshA-decorated pretreated Lm. fermentum cells tolerates gastrointestinal stress, such as low pH and presence of bile acid. To our knowledge, this study is the first to characterize and demonstrate the cell-surface display ability of CshA. The potential application of CshA in non-GMO antigen delivery system and enzyme immobilization remains to be tested.


Assuntos
Hidrolases , Lactobacillus fermentum , Membrana Celular/metabolismo , Parede Celular/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Proteínas de Membrana/metabolismo
6.
Sci Rep ; 12(1): 13546, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941360

RESUMO

Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of Tyrosine (Tyr) degradation pathway essential to animals and the deficiency of FAH causes an inborn lethal disease. In plants, a role of this pathway was unknown until we found that mutation of Short-day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short day. Phenylalanine (Phe) could be converted to Tyr and then degraded in both animals and plants. Phe ingestion in animals worsens the disease caused by FAH defect. However, in this study we found that Phe represses cell death caused by FAH defect in plants. Phe treatment promoted chlorophyll biosynthesis and suppressed the up-regulation of reactive oxygen species marker genes in the sscd1 mutant. Furthermore, the repression of sscd1 cell death by Phe could be reduced by α-aminooxi-ß-phenylpropionic acid but increased by methyl jasmonate, which inhibits or activates Phe ammonia-lyase catalyzing the first step of phenylpropanoid pathway, respectively. In addition, we found that jasmonate signaling up-regulates Phe ammonia-lyase 1 and mediates the methyl jasmonate enhanced repression of sscd1 cell death by Phe. These results uncovered the relation between chlorophyll biosynthesis, phenylpropanoid pathway and jasmonate signaling in regulating the cell death resulting from loss of FAH in plants.


Assuntos
Amônia-Liases , Arabidopsis , Amônia-Liases/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Morte Celular , Clorofila/metabolismo , Hidrolases/metabolismo , Fenilalanina/metabolismo , Tirosina/metabolismo , Tirosina Transaminase/metabolismo
7.
Biochem Biophys Res Commun ; 626: 100-106, 2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-35981419

RESUMO

Polyethylene terephthalate (PET) is one of the most abundantly produced synthetic polyesters. The vast number of waste plastics including PET has challenged the waste management sector while also posing a serious threat to the environment due to improper littering. Recently, enzymatic PET degradation has been shown to be a viable option for a circular plastic economy, which can mitigate the plastic pollution. While protein engineering studies on specific PET degradation enzymes such as leaf-branch compost cutinase (LCC), Thermobifida sp. cutinases and Ideonella sakaiensis PETase (IsPETase) have been extensively published, other homologous PET degrading enzymes have received less attention. Ple629 is a polyester hydrolase identified from marine microbial consortium having activity on PET and the bioplastic polybutylene adipate terephthalate (PBAT). In order to explore its catalytic mechanism and improve its potential for PET hydrolysis, we solved its crystal structure in complex with a PET monomer analogue, and validated its structural and mechanistic similarity to known PET hydrolases. By structural comparisons, we identified some hot spot positions described in previous research on protein engineering of PET hydrolases. We substitute these amino acid residues in Ple629, and obtained variants with improved activity and thermo-stability. The most promising variant D226A/S279A exhibited a more than 5.5-fold improved activity on PET nanoparticles than the wild-type enzyme, suggesting its potential applicability in the biotechnological plastic recycling.


Assuntos
Hidrolases , Plásticos , Hidrolases/metabolismo , Hidrólise , Plásticos/química , Polietilenotereftalatos/metabolismo , Engenharia de Proteínas
8.
Fish Shellfish Immunol ; 128: 67-73, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35921931

RESUMO

The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes.


Assuntos
Saponinas , Pepinos-do-Mar , Stichopus , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Difosfatos , Hidrolases/genética , Hidrolases/metabolismo , Imunidade Inata/genética , Ácido Mevalônico/análogos & derivados , Nucleosídeos , Fosfotransferases (Aceptor do Grupo Fosfato) , Filogenia , Sinais Direcionadores de Proteínas/genética , Pepinos-do-Mar/genética , Espectrometria de Massas em Tandem
9.
Artigo em Inglês | MEDLINE | ID: mdl-35886575

RESUMO

Psoriasis is an autoimmune disease in which the disturbed dependencies between lymphocytes, dendritic cells, keratinocytes and neutrophils play the most important role. One of them is the overproduction of neutrophil extracellular traps (NETs). The release of NETs can be induced by pathogens, as well as antibodies and immune complexes, cytokines and chemokines, including TNFα. The first step of the NET creation is the activation of peptidyl arginine deiminase 4 (PAD-4). PAD-4 seems to be responsible for citrullination of histones and chromatin decondensation, but the data on PAD-4 in NETs is inconclusive. Thus, the current study aimed to determine PAD-4 and TNFα levels in the serum of psoriatic patients by ELISA and observe the response of these factors to systemic (anti-17a, anti-TNFα and methotrexate) therapies. Increased levels of both PAD-4 and its main stimulus factor TNFα in pre-treatment patients have been reported along with the concentrations of proteins correlated with disease severity (PASI, BSA). Before treatment, the irregularities in the case of anti-nuclear antibodies level (ANA) were also observed. All of the applied therapies led to a decrease in PAD-4 and TNFα levels after 12 weeks. The most significant changes, both in protein concentrations as well as in scale scores, were noted with anti-TNFα therapy (adalimumab and infliximab). This phenomenon may be associated with the inhibition of TNFα production at different stages of psoriasis development, including NET creation. The obtained data suggest the participation of PAD-4 in the activation of neutrophils to produce NETs in psoriasis, which may create opportunities for modern therapies with PAD inhibitors. However, further exploration of gene and protein expression in psoriatic skin is needed.


Assuntos
Armadilhas Extracelulares , Proteína-Arginina Desiminase do Tipo 4 , Psoríase , Fator de Necrose Tumoral alfa , Armadilhas Extracelulares/metabolismo , Humanos , Hidrolases/metabolismo , Neutrófilos/metabolismo , Proteína-Arginina Desiminase do Tipo 4/sangue , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Psoríase/tratamento farmacológico , Psoríase/metabolismo , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo
10.
J Hazard Mater ; 438: 129517, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35809363

RESUMO

Accumulation of polyethylene terephthalate (PET) has brought an enormous threat to the ecosystem. The recently reported PET hydrolase (DuraPETase) and MHET hydrolase (MHETase) can synergistically catalyze the complete PET degradation. Hence, this work was designed to develop a bienzymatic cascade catalysis by co-immobilizing the two enzymes for PET biodegradation. DuraPETase and MHETase were sequentially co-immobilized in calcium phosphate nanocrystals (CaP) through SpyTag/SpyCatcher system. MHETase-SpyCatcher was first embedded inside the nanocrystals via biomimetic mineralization, and DuraPETase-SpyTag was then conjugated on the outlayer (~1.5 µm). The bienzyme compartmentalization facilitated DuraPETase interaction with the solid substrate, and the layered structures of the nanocrystals protected the enzymes, thus enhancing their stability. The high specific surface area of the nanocrystals and the proximity effects from the bienzymatic cascade were beneficial to the improved enzyme activity. Experimental data and molecular dynamics simulations revealed the activation effect of Ca2+ on DuraPETase. Taken together, the final results indicate that the PET degradation efficiency of DuraPETase-MHETase@CaP increased by 6.1 and 1.5 times over the free bienzyme system within 10 d at 40 °C and 50 °C, with weight losses at 32.2% and 50.3%, respectively. The bienzymatic cascade with DuraPETase-MHETase@CaP can completely degrade PET, contributing to the recycling of PET.


Assuntos
Nanopartículas , Polietilenotereftalatos , Fosfatos de Cálcio , Ecossistema , Hidrolases/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo
11.
Int J Mol Sci ; 23(14)2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35887122

RESUMO

Recently it was shown that a specific form of male infertility in Holstein cattle was caused by a nonsense variant in the α/ß-hydrolase domain-containing 16B (ABHD16B) gene resulting in a protein truncation at amino acid position 218 (p.218Q*) and loss of function. Lipidomics showed that the absence of ABHD16B influenced the content of phosphatidylcholine (PC), ceramide (Cer), diacylglycerol (DAG), and sphingomyelin (SM) in variant carrier sperm membranes. However, the exact cause of infertility in affected sires has remained unclear until now. To elucidate the cause of infertility, we analyzed (i) standard sperm parameters (i.e., total sperm number, morphological intact sperm, total sperm motility), (ii) in vitro fertilizability and effects on early embryonic development, and (iii) sperm survival rates (i.e., capacitation time). The affected spermatozoa showed no changes in the usual sperm parameters and were also capable of fertilization in vitro. Furthermore, the absence of ABHD16B did not affect early embryonic development. Based on these results, it was concluded that the affected spermatozoa appeared to be fertilizable per se. Consequently, the actual cause of the inability to fertilize could only be due to a time- and/or place-dependent process after artificial insemination and before fertilization. A process fundamental to the ability to fertilize after insemination is capacitation. Capacitation is a biochemical maturation process that spermatozoa undergo in the female genital tract and is inevitable for the successful fertilization of the oocyte. It is known that the presence and concentration of certain sperm membrane lipids are essential for the correct course of capacitation. However, precisely these lipids are absent in the membrane of spermatozoa affected by the ABHD16B truncation. Since all other causes of fertilization inability were excluded in the previous experiments, consequently, the only remaining hypothesis was that the loss of function of ABHD16B leads to a capacitation disruption. We were able to show that heterozygous and homozygous affected spermatozoa exhibit premature capacitation and therefore decay before fertilization. This effect of the loss of function of ABHD16B has not been described before and our studies now revealed why sires harboring the variant in the ABHD16B gene are infertile.


Assuntos
Infertilidade Masculina , Capacitação Espermática , Animais , Bovinos , Feminino , Humanos , Hidrolases/metabolismo , Infertilidade Masculina/metabolismo , Masculino , Sêmen , Motilidade Espermática/genética , Espermatozoides/metabolismo
12.
Int J Mol Sci ; 23(14)2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35887168

RESUMO

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a pivotal enzyme in tocopherol and plastoquinone synthesis and a potential target for novel herbicides. Thirty-five pyridine derivatives were selected to establish a Topomer comparative molecular field analysis (Topomer CoMFA) model to obtain correlation information between HPPD inhibitory activity and the molecular structure. A credible and predictive Topomer CoMFA model was established by "split in two R-groups" cutting methods and fragment combinations (q2 = 0.703, r2 = 0.957, ONC = 6). The established model was used to screen out more active compounds and was optimized through the auto in silico ligand directing evolution (AILDE) platform to obtain potential HPPD inhibitors. Twenty-two new compounds with theoretically good HPPD inhibition were obtained by combining the high-activity contribution substituents in the existing molecules with the R-group search via Topomer search. Molecular docking results revealed that most of the 22 fresh compounds could form stable π-π interactions. The absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction and drug-like properties made 9 compounds potential HPPD inhibitors. Molecular dynamics simulation indicated that Compounds Y12 and Y14 showed good root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values and stability. According to the AILDE online verification, 5 new compounds with potential HPPD inhibition were discovered as HPPD inhibitor candidates. This study provides beneficial insights for subsequent HPPD inhibitor design.


Assuntos
4-Hidroxifenilpiruvato Dioxigenase , Herbicidas , Computadores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Herbicidas/química , Herbicidas/farmacologia , Hidrolases/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular
13.
Angew Chem Int Ed Engl ; 61(37): e202205790, 2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-35856897

RESUMO

We report the discovery of an unusual halohydrin dehalogenase, HHDHamb, that can work under relatively low acidic conditions and extremely low temperatures for the bio-nitration of epoxides using nitrite as a nitrating agent. The bio-nitration strategy exhibits high chemo-, regio-, and enantioselectivity, catalyzing the kinetic resolution of various epoxides to enantiopure ß-nitroalcohols with nitro-bearing stereocenters in up to 41 % isolated yield and >99 % enantiomeric excess (ee). Additionally, the bio-nitration method displays a high reaction efficiency and can be performed on a gram scale. We also solved the crystal structure of HHDHamb to understand the possible structural determinants of chemoselectivity control in the bio-nitration reaction.


Assuntos
Compostos de Epóxi , Hidrolases , Compostos de Epóxi/química , Hidrolases/metabolismo , Cinética , Estereoisomerismo
14.
Animal ; 16(7): 100576, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35777297

RESUMO

Lignification of cellulose limits the effective utilisation of fibre in plant cell wall. Lignocellulose-degrading bacteria secrete enzymes that decompose lignin and have the potential to improve fibre digestibility. Therefore, this study aimed to investigate the effect of whole-plant corn silage inoculated with lignocellulose-degrading bacteria on the growth performance, rumen fermentation, and rumen microbiome in sheep. Twelve 2-month-old male hybrid sheep (Dorper ♂ × small-tailed Han ♀) were randomly assigned into two dietary groups (n = 6): (1) untreated whole-plant corn silage (WPCS) and (2) WPCS inoculated with bacterial inoculant (WPCSB). Whole-plant corn silage inoculated with bacterial inoculant had higher in situ NDF digestibility than WPCS. Sheep in the WPCSB group had significantly higher average daily gain, DM intake, and feed conversion rate than those in the WPCS group (P < 0.05). Furthermore, higher volatile fatty acid concentrations were detected in WPCSB rumen samples, leading to lower ruminal pH (P < 0.05). The WPCSB group showed higher abundance of Bacteroidetes and lower abundance of Firmicutes in the rumen microbiome than the WPCS group (P < 0.05). Multiple differential genera were identified, with Prevotella being the most dominant genus and more abundant in WPCSB samples. Moreover, the enriched functional attributes, including those associated with glycolysis/gluconeogenesis and citrate cycle, were more actively expressed in the WPCSB samples than in the WPCS samples. Additionally, certain glucoside hydrolases that hydrolyse the side chains of hemicelluloses and pectins were also actively expressed in the WPCSB microbiome. These findings suggested that WPCSB increased NDF digestibility in three ways: (1) by increasing the relative abundance of the most abundant genera, (2) by recruiting more functional features involved in glycolysis/gluconeogenesis and citrate cycle pathways, and (3) by increasing the relative abundance and/or expression activity of the glucoside hydrolases involved in hemicellulose and pectin metabolism. Our findings provide novel insights into the microbial mechanisms underlying improvement in the growth performance of sheep/ruminants. However, the biological mechanisms cannot be fully elucidated using only metagenomics tools; therefore, a combined multi-omics approach will be used in subsequent studies.


Assuntos
Microbioma Gastrointestinal , Silagem , Animais , Bactérias/metabolismo , Citratos/farmacologia , Dieta/veterinária , Fibras na Dieta/metabolismo , Digestão , Fermentação , Glucosídeos/farmacologia , Hidrolases/metabolismo , Hidrolases/farmacologia , Lignina/metabolismo , Masculino , Rúmen/metabolismo , Ovinos , Silagem/análise , Zea mays/química
15.
Sci Total Environ ; 846: 157358, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-35850328

RESUMO

There has been a growing interest in poly(ethylene terephthalate) PET degradation studies in the last few years due to its widespread use and large-scale plastic waste accumulation in the environment. One of the most promising enzymatic methods in the context of PET degradation is the use of PETase from Ideonella sakaiensis, which has been reported to be an efficient enzyme for hydrolysing ester bonds in PET. In our study, we expressed a codon-optimized PETase gene in the yeast Yarrowia lipolytica. The obtained strain was tested for its ability to degrade PET directly in culture, and a screening of different supplements that might raise the level of PET hydrolysis was performed. We also carried out long-term cultures with PET film, the surface of which was examined by scanning electron microscopy. The efficiency of PET degradation was tested based on the concentration of degradation products released, and the results showed that supplementation of the culture with olive oil resulted in 66 % higher release of terephthalic acid into the medium compared to the mutant culture without supplementation. The results indicate the possibility of ethylene glycol uptake by both strains, and, additionally, the PETase produced by the newly engineered strain hydrolyses MHET. The structure of the PET film after culture with the modified strain, meanwhile, had numerous surface defects, cracks, and deformations.


Assuntos
Polietilenotereftalatos , Yarrowia , Etilenos , Hidrolases/química , Hidrolases/genética , Hidrolases/metabolismo , Ácidos Ftálicos , Polietilenotereftalatos/química , Yarrowia/genética
16.
Res Vet Sci ; 150: 22-32, 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-35803003

RESUMO

In this study, the complete proteome of goat ejaculated semen including spermatozoa and seminal plasma was established, applying a tandem mass tag (TMT) labeling together with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In seminal plasma, 2299 proteins were identified and 2098 proteins were quantified. The GO analysis demonstrated that 32% proteins were involved in metabolic activities. 46% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. Regarding molecular function, 40% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis indicated a primary involvement of the identified proteins in protein processing in endoplasmic reticulum, lysosome, and proteome. In spermatozoa, 2491 proteins were identified and quantified. 39% proteins are involved in metabolic activities. 48% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. 38% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis demonstrated their roles derived from the identified proteins in proteasome, glycolysis, pyruvate metabolism, and citrate cycle. Additionally, 1312 proteins were simultaneously presented in spermatozoa and seminal plasma. The involvement of 42% proteins in metabolic activities were observed. 47% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. The common proteins are mainly engaged on protein processing in endoplasmic reticulum, proteome, glycolysis, lysosome, and citrate cycle. Collectively, this study established the protein database of goat semen. More studies should be used to elucidate functionality of these identified proteins.


Assuntos
Proteômica , Sêmen , Animais , Cromatografia Líquida/veterinária , Citratos/análise , Citratos/metabolismo , Cabras/metabolismo , Hidrolases/análise , Hidrolases/metabolismo , Masculino , Proteoma , Proteômica/métodos , Espermatozoides/metabolismo , Espectrometria de Massas em Tandem/veterinária
17.
J Biosci Bioeng ; 134(3): 187-194, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35780071

RESUMO

Fucoidans are hetero-sulfated polysaccharides that are widely distributed in brown algae and have been extensively studied for their various biological activities. The structure-function relationship of fucoidans remains unclear but can be studied using fucoidan-degrading enzymes (fucoidanases). Here, we isolated and identified Flavobacterium sp. SW as a microbial strain that can grow on fucoidan from Cladosiphon okamuranus as the sole carbon source. Genomic analysis of this strain revealed the presence of two genes, swfct and swfcn2, that are homologous to fct114 from Luteolibacter algae H18 and fcnA from Psychromonas sp. SW5A, respectively. The gene products were produced in Escherichia coli and showed significantly different specificities for fucoidan. Swfct catalyzed the degradation of deacetylated fucoidan from C. okamuranus, and Swfcn2 degraded fucoidans from Saccharina sculpera and Macrocystis pyrifera. The general properties of Swfct were examined by measuring the amounts of reducing ends produced by the enzymatic reaction, and the enzyme properties of Swfcn2 were evaluated by carbohydrate-polyacrylamide gel electrophoresis. Our findings indicate that one microbial strain can harbor genes encoding two different types of fucoidanases.


Assuntos
Flavobacterium , Feófitas , Flavobacterium/genética , Flavobacterium/metabolismo , Genoma , Hidrolases/metabolismo , Feófitas/genética , Feófitas/metabolismo , Polissacarídeos/metabolismo , Sulfatos/metabolismo
18.
Acta Crystallogr D Struct Biol ; 78(Pt 7): 865-882, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35775986

RESUMO

The mesophilic cyanobacterium Synechocystis sp. PCC 6803 encodes an S-adenosyl-L-homocysteine hydrolase (SAHase) of archaeal origin in its genome. SAHases are essential enzymes involved in the regulation of cellular S-adenosyl-L-methionine (SAM)-dependent methylation reactions. They are usually active as homotetramers or, less commonly, as homodimers. A SAHase subunit is composed of two major domains: a cofactor (NAD+)-binding domain and a substrate (S-adenosyl-L-homocysteine)-binding domain. These are connected by a hinge element that is also a coordination site for an alkali-metal cation that influences domain movement during the catalytic cycle. Typically, the highest activity and strongest substrate binding of bacterial SAHases are observed in the presence of K+ ions. The SAHase from Synechocystis (SynSAHase) is an exception in this respect. Enzymatic and isothermal titration calorimetry studies demonstrated that in contrast to K+-dependent SAHases, the activity and ligand binding of SynSAHase are not affected by the presence of any particular alkali ion. Moreover, in contrast to other SAHases, the cyanobacterial enzyme is in an equilibrium of two distinct oligomeric states corresponding to its dimeric and tetrameric forms in solution. To explain these phenomena, crystal structures of SynSAHase were determined for the enzyme crystallized in the presence of adenosine (a reaction byproduct or substrate) and sodium or rubidium cations. The structural data confirm that while SynSAHase shares common structural features with other SAHases, no alkali metal is coordinated by the cyanobacterial enzyme as a result of a different organization of the macromolecular environment of the site that is normally supposed to coordinate the metal cation. This inspired the generation of SynSAHase mutants that bind alkali-metal cations analogously to K+-dependent SAHases, as confirmed by crystallographic studies. Structural comparisons of the crystal structure of SynSAHase with other experimental models of SAHases suggest a possible explanation for the occurrence of the cyanobacterial enzyme in the tetrameric state. On the other hand, the reason for the existence of SynSAHase in the dimeric state in solution remains elusive.


Assuntos
Hidrolases , Synechocystis , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Hidrolases/química , Hidrolases/metabolismo , Rubídio , S-Adenosilmetionina/metabolismo , Synechocystis/química , Synechocystis/metabolismo
19.
J Biol Chem ; 298(7): 102142, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35714769

RESUMO

The bacterial stringent response involves wide-ranging metabolic reprogramming aimed at increasing long-term survivability during stress conditions. One of the hallmarks of the stringent response is the production of a set of modified nucleotides, known as alarmones, which affect a multitude of cellular pathways in diverse ways. Production and degradation of these molecules depend on the activity of enzymes from the RelA/SpoT homologous family, which come in both bifunctional (containing domains to both synthesize and hydrolyze alarmones) and monofunctional (consisting of only synthetase or hydrolase domain) variants, of which the structure, activity, and regulation of the bifunctional RelA/SpoT homologs have been studied most intensely. Despite playing an important role in guanosine nucleotide homeostasis in particular, mechanisms of regulation of the small alarmone hydrolases (SAHs) are still rather unclear. Here, we present crystal structures of SAH enzymes from Corynebacterium glutamicum (RelHCg) and Leptospira levettii (RelHLl) and show that while being highly similar, structural differences in substrate access and dimer conformations might be important for regulating their activity. We propose that a varied dimer form is a general property of the SAH family, based on current structural information as well as prediction models for this class of enzymes. Finally, subtle structural variations between monofunctional and bifunctional enzymes point to how these different classes of enzymes are regulated.


Assuntos
Guanosina Pentafosfato , Hidrolases , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidrolases/genética , Hidrolases/metabolismo , Ligases/metabolismo , Nucleotídeos/metabolismo
20.
Nat Commun ; 13(1): 3335, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35680936

RESUMO

The Madagascar's periwinkle is the model plant for studies of plant specialized metabolism and monoterpenoid indole alkaloids (MIAs), and an important source for the anticancer medicine vinblastine. The elucidation of entire 28-step biosynthesis of vinblastine allowed further investigations for the formation of other remarkably complex bioactive MIAs. In this study, we describe the discovery and characterization of vindolinine synthase, a Fe(II)/α-ketoglutarate-dependent (Fe/2OG) dioxygenase, that diverts assembly of tabersonine to vinblastine toward the formation of three alternatively cyclized MIAs: 19S-vindolinine, 19R-vindolinine, and venalstonine. Vindolinine synthase catalyzes a highly unusual, redox-neutral reaction to form a radical from dehydrosecodine, which is further cyclized by hydrolase 2 to form the three MIA isomers. We further show the biosynthesis of vindolinine epimers from tabersonine using hydrolase 2 catalyzed reverse cycloaddition. While the occurrence of vindolinines is rare in nature, the more widely found venalstonine derivatives are likely formed from similar redox-neutral reactions by homologous Fe/2OG dioxygenases.


Assuntos
Catharanthus , Alcaloides de Triptamina e Secologanina , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Catharanthus/metabolismo , Compostos Ferrosos/metabolismo , Regulação da Expressão Gênica de Plantas , Hidrolases/metabolismo , Oxirredução , Proteínas de Plantas/genética , Alcaloides de Triptamina e Secologanina/metabolismo , Vimblastina/análogos & derivados , Vimblastina/metabolismo
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