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1.
Science ; 369(6507): 1094-1098, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855335

RESUMO

Bacterial production of gaseous hydrocarbons such as ethylene and methane affects soil environments and atmospheric climate. We demonstrate that biogenic methane and ethylene from terrestrial and freshwater bacteria are directly produced by a previously unknown methionine biosynthesis pathway. This pathway, present in numerous species, uses a nitrogenase-like reductase that is distinct from known nitrogenases and nitrogenase-like reductases and specifically functions in C-S bond breakage to reduce ubiquitous and appreciable volatile organic sulfur compounds such as dimethyl sulfide and (2-methylthio)ethanol. Liberated methanethiol serves as the immediate precursor to methionine, while ethylene or methane is released into the environment. Anaerobic ethylene production by this pathway apparently explains the long-standing observation of ethylene accumulation in oxygen-depleted soils. Methane production reveals an additional bacterial pathway distinct from archaeal methanogenesis.


Assuntos
Proteínas de Bactérias/química , Etilenos/biossíntese , Metano/biossíntese , Metionina/biossíntese , Oxirredutases/química , Rhodospirillum rubrum/enzimologia , Anaerobiose , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biocatálise , Vias Biossintéticas , Oxirredutases/classificação , Oxirredutases/genética , Microbiologia do Solo
2.
Science ; 369(6499): 59-64, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32631887

RESUMO

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.


Assuntos
Cobre/metabolismo , Histonas/química , Oxirredutases/química , Multimerização Proteica , Animais , Biocatálise , Domínio Catalítico/genética , Mutação com Ganho de Função , Histonas/genética , Histonas/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/química , Fatores de Transcrição/metabolismo , Xenopus laevis
3.
Food Chem ; 331: 127352, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32652343

RESUMO

The influence of some additives, including metal ions, antioxidants, enzyme inhibitors and organic solvents, on the storage stability of four organophosphorus pesticides in cucumber samples were investigated. It was found that metal ions, including Al3+, Fe3+, and Co2+, increased the stability of dichlorvos, malathion, and chlorpyrifos. Conversely, Al3+, Fe3+, Fe2+, and Co2+ caused catalytic degradation of diazinon. With the addition of organic solvents (CH2Cl2, CHCl3, CCl4, CH3OH and CH3COCH3), remaining of diazinon residues was higher (16-54%) after storage for seven days. CCl4 was associated with the highest retention of malathion, diazinon, and chlorpyrifos (33%, 48% and 44%, respectively) in samples. SDS also stabilized the pesticides since residues were, again, higher (13-38%) after seven days storage. Furthermore, addition of Al3+ and Fe3+ decreased peroxidase (POD) activity and inhibited degradation of dichlorvos and malathion. After 14 days, lyophilization increased the pesticide residues remaining by 36%, 29%, and 58% for diazinon, malathion and chlorpyrifos, respectively. Overall, the stability of these pesticides during storage is impacted by water content and addition of exogenous substances. This could ensure higher quality of pesticide residue data in samples.


Assuntos
Cucumis sativus/química , Contaminação de Alimentos/análise , Armazenamento de Alimentos , Inseticidas/química , Compostos Organofosforados/química , Alumínio/química , Clorpirifos/análise , Clorpirifos/química , Diazinon/análise , Diazinon/química , Diclorvós/análise , Diclorvós/química , Aditivos Alimentares/química , Liofilização , Inseticidas/análise , Malation/análise , Malation/química , Compostos Organofosforados/análise , Oxirredutases/química , Peroxidases/química , Peroxidases/metabolismo , Resíduos de Praguicidas/análise , Resíduos de Praguicidas/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Dodecilsulfato de Sódio/química , Água/química
4.
Food Chem ; 333: 127492, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659673

RESUMO

In this study, recombinant rice quiescin sulfhydryl oxidase (rQSOX) was expressed and characterized, and its performance in flour-processing quality was further evaluated. The purified rQSOX exhibited the highest sulfhydryl oxidation activity (1.96 IU/mg) using dithiothreitol as a substrate, accompanying the production of H2O2. The optimal temperature and pH were 60 °C and pH 8.0 for rQSOX catalyzing oxidation of dithiothreitol. And rQSOX retained 50% of its maximum activity after incubation at 80 °C for 1 h. Moreover, rQSOX supplementation improved the farinograph properties of dough, indicated by the increased dough stability time and decreased degree of softening, and enhanced viscoelastic properties of the dough. Addition of rQSOX (10 IU/g flour) provided remarkable improvement in specific volume (37%) and springiness (17%) of the steamed bread, and significantly reduced the hardness by half, which was attributed to the strengthened gluten network. The results provide an understanding for rQSOX using in flour-processing industry.


Assuntos
Farinha/análise , Oryza/enzimologia , Oxirredutases/química , Proteínas de Plantas/química , Triticum/química , Biocatálise , Pão/análise , Manipulação de Alimentos , Glutens/química , Dureza , Peróxido de Hidrogênio/análise , Oryza/química , Oryza/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
5.
Food Chem ; 331: 127090, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32593035

RESUMO

Chlorothalonil is a class of 2B carcinogen which is widely used in the prevention and treatment of fungal diseases in food samples. Its residual problem has been increasingly concerned by society. In this paper, a fast and simple colorimetric assay based on Manganese dioxide nanosheets (MnO2 NSs)-oxidize 3,3',5,5'-tetramethylbenzidine (TMB) platform was used to detect residual pesticide chlorothalonil in food samples. Under optimal conditions, the half maximal inhibitory concentration and the limit of detection of chlorothalonil were 3.27 and 0.024 ng/mL. There were no obvious cross-reactivity between chlorothalonil and interference substances. The recoveries shown the satisfactory results. The results of colorimetric assay for the authentic samples were largely consistent with gas chromatography. Therefore, the proposed method would be convenient and satisfactory analytical methods for the monitoring of chlorothalonil. Furthermore, the MnO2 - TMB system was used to produce test strips for quick and convenient visual detection of chlorothalonil with good performance.


Assuntos
Colorimetria/métodos , Análise de Alimentos/métodos , Compostos de Manganês/química , Nanoestruturas/química , Nitrilos/análise , Óxidos/química , Benzidinas/química , Colorimetria/instrumentação , Análise de Alimentos/instrumentação , Contaminação de Alimentos/análise , Fungicidas Industriais/análise , Limite de Detecção , Oxirredução , Oxirredutases/química
6.
Proc Natl Acad Sci U S A ; 117(17): 9349-9355, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291342

RESUMO

Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome c oxidase (CcO). CcO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H+/e- stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CcO's hydrophobic core and on whether bacterial and mitochondrial CcOs operate via the same coupling mechanism. To resolve this, we exploited the unique amenability to mitochondrial DNA mutagenesis of the yeast Saccharomyces cerevisiae to introduce single point mutations in the hydrophilic pathways of CcO to test function. From adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria, we definitely established that the D-channel, and not the H-channel, is the proton pump of the yeast mitochondrial enzyme, supporting an identical coupling mechanism in all forms of the enzyme.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Oxirredutases/química , Bactérias/metabolismo , Cobre/química , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Íons , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Oxigênio/metabolismo , Bombas de Próton/metabolismo , Prótons , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Chemistry ; 26(22): 4884, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32297426

RESUMO

Invited for the cover of this issue is the group of Ulrich Schwaneberg at the Institute of Biotechnology, RWTH-Aachen University and DWI Lebniz Institute of Interactive Materials. The picture calls for special attention to be paid to the extra Cu binding site of Copper efflux Oxidase (CueO), due to its predominant function in tuning the electrocatalytic kinetics towards oxygen reduction. Read the full text of the article at 10.1002/chem.201905598.


Assuntos
Ceruloplasmina/metabolismo , Lacase/química , Oxirredutases/química , Sítios de Ligação , Ceruloplasmina/química , Transporte de Elétrons , Proteínas de Escherichia coli/química , Cinética , Oxirredução , Oxirredutases/metabolismo
8.
Nat Commun ; 11(1): 1454, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193396

RESUMO

Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis are arising in the field of deuterium labelling, where compounds bearing deuterium (2H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for 2H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4-2H]-NADH). Here we report on a strategy that combines a clean reductant (H2) with a cheap source of 2H-atoms (2H2O) to generate and recycle [4-2H]-NADH. By coupling [4-2H]-NADH-recycling to an array of C=O, C=N, and C=C bond reductases, we demonstrate asymmetric deuteration across a range of organic molecules under ambient conditions with near-perfect chemo-, stereo- and isotopic selectivity. We demonstrate the synthetic utility of the system by applying it in the isolation of the heavy drug (1S,3'R)-[2',2',3'-2H3]-solifenacin fumarate on a preparative scale.


Assuntos
Biocatálise , Técnicas de Química Sintética/métodos , Deutério/química , Marcação por Isótopo/métodos , Oxirredutases/química , Óxido de Deutério/química , Estrutura Molecular , Niacinamida/química , Succinato de Solifenacina/química , Estereoisomerismo
9.
Nat Commun ; 11(1): 864, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054833

RESUMO

Siroheme is the central cofactor in a conserved class of sulfite and nitrite reductases that catalyze the six-electron reduction of sulfite to sulfide and nitrite to ammonia. In Salmonella enterica serovar Typhimurium, siroheme is produced by a trifunctional enzyme, siroheme synthase (CysG). A bifunctional active site that is distinct from its methyltransferase activity catalyzes the final two steps, NAD+-dependent dehydrogenation and iron chelation. How this active site performs such different chemistries is unknown. Here, we report the structures of CysG bound to precorrin-2, the initial substrate; sirohydrochlorin, the dehydrogenation product/chelation substrate; and a cobalt-sirohydrochlorin product. We identified binding poses for all three tetrapyrroles and tested the roles of specific amino acids in both activities to give insights into how a bifunctional active site catalyzes two different chemistries and acts as an iron-specific chelatase in the final step of siroheme synthesis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Heme/análogos & derivados , Metiltransferases/química , Metiltransferases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Eletroquímica , Ferroquelatase/química , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/biossíntese , Heme/química , Metiltransferases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidade por Substrato , Tetrapirróis/química , Tetrapirróis/metabolismo , Uroporfirinas/química , Uroporfirinas/metabolismo
10.
Nat Commun ; 11(1): 595, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001716

RESUMO

Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients' primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy.


Assuntos
Epilepsia/genética , Genes Recessivos , Mutação com Perda de Função/genética , Oxirredutases/genética , Uridina Difosfato Glucose Desidrogenase/genética , Adolescente , Alelos , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Cinética , Masculino , Organoides/patologia , Oxirredutases/química , Linhagem , Domínios Proteicos , Síndrome , Peixe-Zebra
11.
Chem Commun (Camb) ; 56(20): 3035-3038, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32048636

RESUMO

It remains challenging to develop new materials exhibiting enzyme-like activities and understand the structure-property correlations and catalytic mechanisms. In this study, the characteristics, mechanisms, and applications of a light-activated mimic oxidase based on semiconducting polymer dots (Pdots) prepared from an organic conjugated polymer are demonstrated.


Assuntos
Luz , Polímeros/química , Pontos Quânticos/química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Catálise , Estrutura Molecular , Oxirredutases/química , Oxirredutases/metabolismo , Semicondutores
12.
PLoS Biol ; 18(2): e3000507, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32092071

RESUMO

The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.


Assuntos
Aminoácidos/metabolismo , Methanosarcina/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Domínio Catalítico , Methanosarcina/genética , Methanosarcina/crescimento & desenvolvimento , Methanosarcina/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Mutação , Óperon , Oxirredutases/genética , Fenótipo , Processamento de Proteína Pós-Traducional/genética , Subunidades Proteicas , Temperatura
13.
Proc Natl Acad Sci U S A ; 117(10): 5280-5290, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32094184

RESUMO

Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.


Assuntos
Proteínas de Bactérias/química , Domínio Catalítico , Cobre/química , Ectothiorhodospiraceae/enzimologia , Oxirredutases/química , Bactérias Redutoras de Enxofre/enzimologia , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Oxirredução , Oxigênio/química , Enxofre/química
14.
Biochim Biophys Acta Bioenerg ; 1861(5-6): 148175, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32061652

RESUMO

Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile318-Met322, Gln338-Asp342, Tyr353-Leu357, and Thr368-Ile372 as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.


Assuntos
Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Alanina/genética , Sequência de Aminoácidos , Membrana Celular/metabolismo , Grupo dos Citocromos b/isolamento & purificação , Complexo de Proteínas da Cadeia de Transporte de Elétrons/isolamento & purificação , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/isolamento & purificação , Heme/metabolismo , Mutagênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Oxirredutases/isolamento & purificação , Consumo de Oxigênio , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
15.
Appl Environ Microbiol ; 86(8)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32033954

RESUMO

Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins.IMPORTANCE Cytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Celulose/metabolismo , Cytophaga/genética , Oxirredutases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cytophaga/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Alinhamento de Sequência
16.
Talanta ; 211: 120707, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32070586

RESUMO

In this study, a colorimetric sensing assay of isoniazid based on excellent oxidase-like activity of heparin sodium stabilized platinum nanoparticles (HS-PtNPs) has been demonstrated. The newly prepared HS-PtNPs exhibit a great dispersion with an average size distribution of 4.8 ± 0.6 nm, and maintain more than 90% catalytic activity under strong acid and alkali or long-term storage conditions, indicating a robust nanomaterial with attractive potential. The HS-PtNPs show distinct oxidase-like activity with an ultrahigh affinity (Km = 0.01012 mM) for 3, 3', 5, 5'-tetramethylbenzidine (TMB). More significantly, we found that the pyridine ring of isoniazid has a strong reductive hydrazyl substitution, which can compete with TMB for the catalytic site of HS-PtNPs resulting in a colorless solution. Accordingly, a colorimetric sensing of isoniazid was fabricated. A linear relationship for isoniazid was achieved in 2.5 × 10-6 to 2.5 × 10-4 M (R2 = 0.998) with a low limit of detection 1.7 × 10-6 M (S/N = 3). Recovery experiments in drug tablets show that the standard recovery rates were 95%-103%. The quantitative detection data for isoniazid in drug tablets calculated respectively from the standard method and this method exhibited a high correlation coefficient (a slope of 0.9995), suggesting that high accuracy in isoniazid detection.


Assuntos
Antituberculosos/análise , Heparina/química , Isoniazida/análise , Nanopartículas Metálicas/química , Platina/química , Antituberculosos/química , Benzidinas/química , Colorimetria , Isoniazida/química , Oxirredutases/química , Comprimidos
17.
Ecotoxicol Environ Saf ; 191: 110186, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31954922

RESUMO

A three-dimensional quantitative structure-activity relationship (3D-QSAR) model was established based on molecular structures and docking scores (representing the biodegradability); the scores were obtained for 23 fluoroquinolones (FQs) and the oxidoreductase (PDB ID: 1YZP) of Phanerochaete chrysosporium in the aerobic process of municipal wastewater treatment plants. In the Comparative Molecular Field Analysis (CoMFA) model, q2 was 0.516 and r2pred was 0.727, which showed that the model was reliable and robust. The modification information obtained by the contour maps showed that introducing electronegative, bulky or electropositive groups at different active sites could increase the biodegradability of fluoroquinolone derivatives. Using levofloxacin (LEV) as a modified molecule, 35 fluoroquinolone derivatives with higher biodegradability than LEV were designed. After the evaluation of genotoxicity, bioconcentration and photodegradation, Derivative-15, with higher biodegradability (increased by 27.85%), higher genotoxicity, higher photodegradation and lower bioconcentration, was identified as the most environmentally friendly fluoroquinolone derivative. The 2D-QSAR model of FQ biodegradability was established through the quantization parameters, and q+ was identified as the main parameter affecting the biodegradability of FQs through sensitivity analysis. In addition, the docking results of LEV and Derivative-15 with the oxidoreductase in P. chrysosporium showed that the electrostatic field force between Derivative-15 and the amino acid residues promoted the binding of the donor to the receptor protein, thereby increasing the biodegradability of Derivative-15. Additionally, molecular dynamics simulations revealed that the enhancement of the electrostatic field force with Derivative-15 could promote the binding of the ligand to the receptor, which was basically consistent with the conclusion of molecular docking. Finally, the three microbial degradation pathways of LEV and Derivative-15 were also proposed. The total energy barrier value of the pathway with the lowest total energy barrier of biodegradation was reduced by 32.07%, which was basically consistent with the enhancement of biodegradability of Derivative-15.


Assuntos
Fluoroquinolonas/química , Modelos Moleculares , Poluentes Químicos da Água/química , Basidiomycota/enzimologia , Biodegradação Ambiental , Fluoroquinolonas/farmacocinética , Fluoroquinolonas/toxicidade , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Oxirredutases/química , Relação Quantitativa Estrutura-Atividade , Poluentes Químicos da Água/farmacocinética , Poluentes Químicos da Água/toxicidade
18.
PLoS One ; 15(1): e0227977, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978122

RESUMO

Anabaena variabilis is a diazotrophic filamentous cyanobacterium that differentiates to heterocysts and produces hydrogen as a byproduct. Study on metabolic interactions of the two differentiated cells provides a better understanding of its metabolism especially for improving hydrogen production. To this end, a genome-scale metabolic model for Anabaena variabilis ATCC 29413, iAM957, was reconstructed and evaluated in this research. Then, the model and transcriptomic data of the vegetative and heterocyst cells were applied to construct a regulated two-cell metabolic model. The regulated model improved prediction for biomass in high radiation levels. The regulated model predicts that heterocysts provide an oxygen-free environment and then, this model was used to find strategies for improving hydrogen production in heterocysts. The predictions indicate that the removal of uptake hydrogenase improves hydrogen production which is consistent with previous empirical research. Furthermore, the regulated model proposed activation of some reactions to provide redox cofactors which are required for improving hydrogen production up to 60% by bidirectional hydrogenase.


Assuntos
Anabaena variabilis/metabolismo , Hidrogênio/metabolismo , Redes e Vias Metabólicas , Oxirredutases/química , Anabaena variabilis/química , Anabaena variabilis/genética , Biocombustíveis , Oxirredutases/metabolismo , Oxigênio/metabolismo , Fótons , Transcriptoma/genética
19.
Nat Commun ; 11(1): 316, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949281

RESUMO

Here we propose an experimental setup based on operando X-ray absorption spectroscopy (XAS) to understand why copper-containing oxidoreductase enzymes show exceptional performance as catalysts for the oxygen reduction reaction (ORR). An electrode based on carbon nanoparticles organized in mesoporous structures with bilirubin oxidase (BOD) was developed to be used in a home-made operando XAS electrochemical cell, and we probed the electron transfer under ORR regime. In the presence of molecular oxygen, the BOD cofactor containing 4 copper ions require an overpotential about 150 mV to be reduced as compared to that in the absence of oxygen. A second electron transfer step, which occurs faster than the cofactor reduction, suggests that the cooper ions act as a tridimensional redox active electronic bridges for the electron transfer reaction.


Assuntos
Cobre/química , Transporte de Elétrons , Elétrons , Metaloproteínas/química , Oxirredutases/química , Espectroscopia por Absorção de Raios X/métodos , Catálise , Eletrodos , Modelos Químicos , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxigênio/química , Saccharomycetales
20.
J Steroid Biochem Mol Biol ; 199: 105586, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31926269

RESUMO

Recent studies have shown that an adrenal steroid 11ß-hydroxy-4-androstene-3,17-dione serves as the precursor to androgens, 11-ketotestosterone and 11-ketodihydrotestosterone (11KDHT). The biosynthetic pathways include the reduction of 3- and 17-keto groups of the androgen precursors 11-keto-C19-steroids, which has been reported to be mediated by three human enzymes; aldo-keto reductase (AKR)1C2, AKR1C3 and 17ß-hydroxysteroid dehydrogenase (HSD) type-3. To explore the contribution of the enzymes in the reductive metabolism, we kinetically compared the substrate specificity for 11-keto-C19-steroids among purified recombinant preparations of four AKRs (1C1, 1C2,1C3 and 1C4) and DHRS11, which shows 17ß-HSD activity. Although AKR1C1 did not reduce the 11-keto-C19-steroids, AKR1C3 and DHRS11 reduced 17-keto groups of 11-keto-4-androstene-3,17-dione, 11-keto-5α-androstane-3,17-dione (11K-Adione) and 11-ketoandrosterone with Km values of 5-28 µM. The 3-keto groups of 11KDHT and 11K-Adione were reduced by AKR1C4 (Km 1 µM) more efficiently than by AKR1C2 (Km 5 and 8 µM, respectively). GC/MS analysis of the products showed that DHRS11 acts as 17ß-HSD, and that AKR1C2 and AKR1C4 are predominantly 3α-HSDs, but formed a minor 3ß-metabolite from 11KDHT. Since DHRS11 was thus newly identified as 11-keto-C19-steroid reductase, we also investigated its substrate-binding mode by molecular docking and site-directed mutagenesis of Thr163 and Val200, and found the following structural features: 1). There is a space that accommodates the 11-keto group of the 11-keto-C19-steroids in the substrate-binding site. 2) Val200 is a critical determinant for exhibiting the strict 17ß-HSD activity of the enzyme, because the Val200Leu mutation resulted in both significant impairment of the 17ß-HSD activity and emergence of 3ß-HSD activity towards 5α-androstanes including 11KDHT.


Assuntos
17-Hidroxiesteroide Desidrogenases/química , 20-Hidroxiesteroide Desidrogenases/química , Aldo-Ceto Redutases/química , Esteroides/biossíntese , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , 20-Hidroxiesteroide Desidrogenases/genética , 20-Hidroxiesteroide Desidrogenases/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/química , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Aldo-Ceto Redutases/genética , Aldo-Ceto Redutases/metabolismo , Androgênios/biossíntese , Androgênios/química , Vias Biossintéticas/genética , Humanos , Simulação de Acoplamento Molecular , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Esteroides/química , Especificidade por Substrato , Testosterona/análogos & derivados , Testosterona/metabolismo
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