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1.
Chemosphere ; 248: 126037, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32018111

RESUMO

This study was focused on gaining insights into the mechanism by which the herbicide- Spectracide®, induces oxidative stress and alters behavior in Drosophila melanogaster. Exposure to Spectracide® (50%) significantly (p < 0.05) reduced the negative geotaxis response, jumping behavior and dampened locomotor activity rhythm in adult flies compared to non-exposed flies. Protein carbonyl levels indicative of oxidative damage increased significantly coupled with down-regulation of Sniffer gene expression encoding carbonyl reductase (CR) and its activity in Spectracide®-exposed flies. In silico modeling analysis revealed that the active ingredients of Spectracide® (atrazine, diquat dibromide, fluazifop-p-butyl, and dicamba) have significant binding affinity to the active site of CR enzyme, with atrazine having comparatively greater affinity. Our results suggest a mechanism by which ingredients in Spectracide® induce oxidative damage by competitive binding to the active site of a protective enzyme and impair its ability to prevent damage to proteins thereby leading to deficits in locomotor behavior in Drosophila.


Assuntos
Herbicidas/toxicidade , Modelos Moleculares , Oxirredutases do Álcool/metabolismo , Animais , Atrazina/toxicidade , Comportamento Animal/efeitos dos fármacos , Drosophila melanogaster/genética , Expressão Gênica , Locomoção/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/genética
2.
Proc Natl Acad Sci U S A ; 117(3): 1485-1495, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31911473

RESUMO

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins' sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins' sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily.


Assuntos
Proteínas de Escherichia coli/química , Proteínas Intrinsicamente Desordenadas/química , Dobramento de Proteína , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Simulação de Dinâmica Molecular , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/metabolismo , Biossíntese de Proteínas , Metiltransferases de Proteína/química , Metiltransferases de Proteína/genética , Metiltransferases de Proteína/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
3.
Appl Biochem Biotechnol ; 190(1): 18-29, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31301008

RESUMO

NAD(P)H-dependent enzymes are ideal biocatalysts for the industrial production of chiral compounds, such as chiral alcohols, chiral amino acids, and chiral amines; however, efficient strategies for the regeneration of coenzyme are expected as costly of the coenzymes. Herein, a solvent-tolerant isopropanol dehydrogenase (IDH) showing lower similarity (37%) with other proteins was obtained and characterized. The enzyme exhibits high catalysis ability of its substrates methanol, ethanol, ethylene glycol, glycerol, isopropanol, n-butanol, isobutanol, and acetone. And it has good adaptability in organic solvents (isopropanol, acetonitrile, acetone, and acetophenone). Interaction force and the corresponding amino acid residues between IDH and NAD+ or NADP+ were parsed by docking. The wide substrate spectrum, excellent organic solvent tolerance, and good biocatalytic activity make the excavated enzyme a promising biocatalyst for the production of chiral compounds industrially and the construction of coenzyme regeneration systems in aqueous organic phase or organic phase.


Assuntos
Oxirredutases do Álcool/metabolismo , Coenzimas/metabolismo , Solventes/metabolismo , Oxirredutases do Álcool/genética , Sítios de Ligação , Clonagem Molecular , Cinética , Simulação de Acoplamento Molecular , NAD/metabolismo , NADP/metabolismo , Compostos Orgânicos/metabolismo , Especificidade por Substrato
4.
J Chem Ecol ; 46(2): 217-231, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31879865

RESUMO

Despite active research, antiherbivore activity of specific plant phenolics remains largely unresolved. We constructed silver birch (Betula pendula) lines with modified phenolic metabolism to study the effects of foliar flavonoids and condensed tannins on consumption and growth of larvae of a generalist herbivore, the autumnal moth (Epirrita autumnata). We conducted a feeding experiment using birch lines in which expression of dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS) or anthocyanidin reductase (ANR) had been decreased by RNA interference. Modification-specific effects on plant phenolics, nutrients and phenotype, and on larval consumption and growth were analyzed using uni- and multivariate methods. Inhibiting DFR expression increased the concentration of flavonoids at the expense of condensed tannins, and silencing DFR and ANR decreased leaf and plant size. E. autumnata larvae consumed on average 82% less of DFRi plants than of unmodified controls, suggesting that flavonoids or glandular trichomes deter larval feeding. However, larval growth efficiency was highest on low-tannin DFRi plants, indicating that condensed tannins (or their monomers) are physiologically more harmful than non-tannin flavonoids for E. autumnata larvae. Our results show that genetic manipulation of the flavonoid pathway in plants can effectively be used to produce altered phenolic profiles required for elucidating the roles of low-molecular weight phenolics and condensed tannins in plant-herbivore relationships, and suggest that phenolic secondary metabolites participate in regulation of plant growth.


Assuntos
Betula/química , Flavonoides/metabolismo , Mariposas/fisiologia , Plantas Geneticamente Modificadas/química , Taninos/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Betula/enzimologia , Betula/parasitologia , Flavonoides/farmacologia , Herbivoria/efeitos dos fármacos , Interações Hospedeiro-Parasita , Larva/crescimento & desenvolvimento , Larva/fisiologia , Mariposas/crescimento & desenvolvimento , Oxigenases/antagonistas & inibidores , Oxigenases/genética , Oxigenases/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Interferência de RNA , Taninos/farmacologia
5.
Enzyme Microb Technol ; 132: 109415, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731965

RESUMO

Aldo-keto reductases (AKRs) are nicotinamide-dependent enzymes that catalyze the transformation of aldehydes and ketones into alcohols. They are spread across all phyla, and those from microbial origin have proved to be highly robust and versatile biocatalysts. In this work, we have discovered and characterized a microbial AKR from the yeast Rhodotorula mucilaginosa by combining genome-mining and expression assays. The new enzyme, named AKR3B4, was expressed by a simple protocol in very good amounts. It displays a selective substrate profile exclusively transforming aldehydes into alcohols. Also, AKR3B4 shows very good stability at medium temperatures, in a broad range of pH values and in the presence of green organic solvents. Conversion assays demonstrate it is an excellent biocatalyst to be used in the synthesis of aromatic alcohols, and also to produce furan-3-ylmethanol and the valuable sweetener xylitol. These results show that AKR3B4 displays attractive features so as to be used in chemoenzymatic processes.


Assuntos
Aldo-Ceto Redutases/genética , Aldo-Ceto Redutases/metabolismo , Rhodotorula/enzimologia , Rhodotorula/genética , Oxirredutases do Álcool/metabolismo , Álcoois/metabolismo , Aldeído Redutase/metabolismo , Aldeídos/metabolismo , Clonagem Molecular , Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Especificidade por Substrato
6.
Virol J ; 16(1): 161, 2019 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-31864392

RESUMO

BACKGROUND: Adenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear. METHODS: The dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding. RESULTS: PMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells. CONCLUSIONS: Our data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation.


Assuntos
Adenoviridae/crescimento & desenvolvimento , Proteínas E1A de Adenovirus/metabolismo , Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Linfócitos/virologia , NAD/metabolismo , Ativação Viral , Adenoviridae/genética , Infecções por Adenovirus Humanos/virologia , Linhagem Celular , Regulação Viral da Expressão Gênica , Humanos , Ativação Linfocitária , Ligação Proteica , Latência Viral
7.
Chem Commun (Camb) ; 55(96): 14462-14465, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31728457

RESUMO

Structure-based engineering of a NAD+-dependent secondary alcohol dehydrogenase from Micrococcus luteus led to a 1800-fold increase in catalytic efficiency for NADP+. Furthermore, the engineered enzymes (e.g., D37S/A38R/V39S/T15I) were successfully coupled to a NADPH-dependent Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440 for redox-neutral biotransformations of C18 fatty acids into C9 chemicals.


Assuntos
Oxirredutases do Álcool/metabolismo , Micrococcus luteus/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Sítios de Ligação , Biotransformação , Ácidos Graxos/metabolismo , Engenharia Genética , Cinética , Oxigenases de Função Mista/metabolismo , Simulação de Dinâmica Molecular , Oxirredução , Pseudomonas/enzimologia
8.
BMC Plant Biol ; 19(1): 476, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694546

RESUMO

BACKGROUND: The outer peels of pomegranate (Punica granatum L.) possess two groups of polyphenols that have health beneficial properties: anthocyanins (ATs, which also affect peel color); and hydrolysable tannins (HTs). Their biosynthesis intersects at 3-dehydroshikimate (3-DHS) in the shikimate pathway by the activity of shikimate dehydrogenase (SDH), which converts 3-DHS to shikimate (providing the precursor for AT biosynthesis) or to gallic acid (the precursor for HTs biosynthesis) using NADPH or NADP+ as a cofactor. The aim of this study is to gain more knowledge about the factors that regulate the levels of HTs and ATs, and the role of SDH. RESULTS: The results have shown that the levels of ATs and HTs are negatively correlated in the outer fruit peels of 33 pomegranate accessions, in the outer peels of two fruits exposed to sunlight, and in those covered by paper bags. When calli obtained from the outer fruit peel were subjected to light/dark treatment and osmotic stresses (imposed by different sucrose concentrations), it was shown that light with high sucrose promotes the synthesis of ATs, while dark at the same sucrose concentration promotes the synthesis of HTs. To verify the role of SDH, six PgSDHs (PgSDH1, PgSDH3-1,2, PgSDH3a-1,2 and PgSDH4) were identified in pomegranate. The expression of PgSDH1, which presumably contributes to shikimate biosynthesis, was relatively constant at different sucrose concentrations. However, the transcript levels of PgSDH3s and PgSDH4 increased with the accumulation of gallic acid and HTs under osmotic stress, which apparently accumulates to protect the cells from the stress. CONCLUSIONS: The results strongly suggest that the biosynthesis of HTs and ATs competes for the same substrate, 3-DHS, and that SDH activity is regulated not only by the NADPH/NADP+ ratio, but also by the expression of the PgSDHs. Since the outer peel affects the customer's decision regarding fruit consumption, such knowledge could be utilized for the development of new genetic markers for breeding pomegranates having higher levels of both ATs and HTs.


Assuntos
Oxirredutases do Álcool/metabolismo , Antocianinas/biossíntese , Taninos Hidrolisáveis/metabolismo , /enzimologia , Frutas/enzimologia
9.
BMC Plant Biol ; 19(1): 434, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31638916

RESUMO

BACKGROUND: Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop's sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. RESULTS: To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40-1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40-1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. CONCLUSIONS: Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40-1 expression, whereas higher miR156 overexpression results in drought susceptibility.


Assuntos
Oxirredutases do Álcool/metabolismo , Medicago sativa/genética , MicroRNAs/genética , Oxirredutases do Álcool/genética , Secas , Regulação da Expressão Gênica de Plantas , Medicago sativa/enzimologia , Medicago sativa/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , RNA de Plantas/genética , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Nat Commun ; 10(1): 4068, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492851

RESUMO

The aldehyde dehydrogenase (ALDH) family of metabolic enzymes converts aldehydes to carboxylates. Here, we find that the reductive consequence of ALDH7A1 activity, which generates NADH (nicotinamide adenine dinucleotide, reduced form) from NAD, underlies how ALDH7A1 coordinates a broad inhibition of the intracellular transport pathways. Studying vesicle formation by the Coat Protein I (COPI) complex, we elucidate that NADH generated by ALDH7A1 targets Brefeldin-A ADP-Ribosylated Substrate (BARS) to inhibit COPI vesicle fission. Moreover, defining a physiologic role for the broad transport inhibition exerted by ALDH7A1, we find that it acts to reduce energy consumption during hypoxia and starvation to promote cellular energy homeostasis. These findings advance the understanding of intracellular transport by revealing how the coordination of multiple pathways can be achieved, and also defining circumstances when such coordination is needed, as well as uncovering an unexpected way that NADH acts in cellular energetics.


Assuntos
Oxirredutases do Álcool/metabolismo , Aldeído Desidrogenase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético , Homeostase , Espaço Intracelular/metabolismo , Oxirredutases do Álcool/genética , Aldeído Desidrogenase/genética , Transporte Biológico , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Hipóxia Celular , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , NAD/metabolismo , Transdução de Sinais , Inanição
11.
Gene ; 716: 144024, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31390541

RESUMO

The young leaves generally accumulate a certain concentration anthocyanins in the dominant species of the subtropical forest, and the changes of anthocyanin synthesis-related enzyme genes expression levels had an important effect on the study photoprotection of anthocyanins in the young leaves of subtropical forests. The determination of anthocyanin synthesis-related enzyme gene sequences and the selection of appropriate reference genes provide a basis for analyzing the functional properties of anthocyanins. In this study, four dominant subtropical forest species (i.e., Schima superba, Castanopsis fissa, Acmena acuminatissima, Cryptocarya concinna) were taken as materials. To obtain the correct nucleotide sequences of anthocyanin-related enzymes, the nucleotide sequences of CHS, DFR and ANS in each dominant species were obtained by sequencing and comparison. Then, to select the most stable reference genes for leaves at different developmental stages and different light conditions, the expression levels of six reference genes, including 18S, Actin, GAPDH, TUB, EF1 and UBQ, were studied by real-time fluorescent quantitative PCR (qRT-PCR), and reference gene stability was analyzed by GeNorm and NormFinder software. The results showed that the expression level of Actin was the most stable in S. superba, A. acuminatissima and C. concinna, and the expression level of GAPDH was the most stable in C. fissa. Finally, the expression levels of the anthocyanin synthesis genes CHS, DFR and ANS were analyzed and found to be consistent with the accumulation trend of anthocyanins in leaves. This study has important theoretical and practical significance for future research into the expression of anthocyanin synthesis-related enzyme genes in the dominant tree species in subtropical forests and reveals that anthocyanin has a photoprotective effect for young leaves in high-light environments.


Assuntos
Antocianinas/biossíntese , Árvores/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Antocianinas/metabolismo , Florestas , Genes de Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismo , RNA de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Alinhamento de Sequência , Análise de Sequência , Árvores/enzimologia , Árvores/metabolismo
12.
Cell Biochem Funct ; 37(7): 534-544, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418900

RESUMO

Chemotherapeutic drugs that induce DNA damage have the potential to kill cancer cells, but DNA repair protects cells from damage-induced cell death. Thus, eliminating DNA repair is a potential approach to overcome cell drug resistance. In this study, we observed that the gene expression of C-terminal binding protein interacting protein (CTIP) was promoted by TNF-α stimulation and prevented TNF-α-induced double-strand breaks (DSBs) in the genomes of cervical cancer cells. The putative miR-130b targeted site within 3' untranslated region (UTR) of CTIP mRNA was identified through in silico analysis and confirmed based on experimental data. By targeting the CTIP gene, miR-130b caused the accumulation of DSBs and accelerated cell apoptosis in combination with poly ADP ribose polymerase (PARP) inhibitors. Additionally, overexpression of the CTIP gene elevated cancer cell viability by promoting proliferation while miR-130b antagonized CTIP-stimulated cell reproduction. Consequently, miR-130b destruction of DNA repair should be employed as a strategy to treat cervical cancer. SIGNIFICANCE OF THE STUDY: Cervical cancer threatens the health of women all over the world. In this study, we observed that miR-130b was able to cause the accumulation of DNA double-strand breaks through suppressing the gene expression of C-terminal binding protein interacting protein and to accelerate cell apoptosis by preventing DNA damage repairs in cervical cancer cells. As far as we know, the impact of miR-130b on the DNA double-strand break repair and on the cell apoptosis induced by the destruction of DNA repair in cervical cancer cells was firstly documented. It is reasonable to believe that miR-130b destruction of DNA repair may be employed as a strategy to treat cervical cancer in the future.


Assuntos
Oxirredutases do Álcool/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , MicroRNAs/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias do Colo do Útero/metabolismo , Reparo do DNA , Feminino , Células HeLa , Humanos , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/patologia
13.
Comput Biol Chem ; 83: 107098, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31421413

RESUMO

Shikimate dehydrogenase (SDH) catalyzes the reversible, NADPH-dependent reduction of 3-dehydroshikimate to shikimate, involved in the shikimate pathway. This pathway has emerged as an important target for the development of antimicrobial agent. Structural and functional analyses suggest that the conserved Lys69 plays an important role in the catalytic activity of Helicobacter pylori (H. pylori) SDH. However, the detailed mechanism how mutation of Lys69 affects the catalytic activity of H. pylori SDH remains unclear. Here, two-layered ONIOM-based quantum mechanics/molecular mechanics (QM/MM) calculation and molecular dynamics (MD) simulations were performed to explore the role of Lys69 in the H. pylori SDH. Our results showed that in addition to act as a catalytic base, the conserved Lys69 plays an additional, important role in the maintenance of the substrate shikimate in the active site, facilitating the catalytic reaction between the cofactor NADP+ and shikimate. Mutation of Lys69 triggers the movement of shikimate away from the active site of SDH, thereby disrupting the catalytic activity. This result can advance our understanding the catalytic mechanism of SDH family, which may benefit of the rational design of SDH inhibitors.


Assuntos
Oxirredutases do Álcool/metabolismo , Helicobacter pylori/enzimologia , Lisina/metabolismo , Simulação de Dinâmica Molecular , Teoria Quântica , Biocatálise , Lisina/química , Lisina/genética
14.
Genes (Basel) ; 10(8)2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31409011

RESUMO

Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3.


Assuntos
Hirudinas/genética , Metanol/metabolismo , Pichia/genética , Transcriptoma , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hirudinas/metabolismo , Pichia/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Redox Biol ; 26: 101301, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31442912

RESUMO

Treatment of tumor cells with H2O2 and nitrite, two long-lived species derived from cold atmospheric plasma, induces a complex autoamplificatory, singlet oxygen-mediated process, which leads to catalase inactivation and reactivation of intercellular apoptosis-inducing signaling. Experimental dissection and quantification of this process is described in this study. When tumor cells were pretreated with H2O2 and nitrite, and then were added to untreated tumor cells, they propaged singlet oxygen mediated catalase inactivation and generation of singlet oxygen to the untreated cell population. This bystander effect allowed to analyze the biochemical requirements of a) induction of the bystander effect-inducing potential, b) transmission of the bystander effect to untreated neighbouring cells, and c) the biochemical consequences of these signaling events. The induction of bystander effect-inducing potential requires the generation of "primary singlet oxygen" through the reactions following the interaction between nitrite and H2O2, followed by local inactivation of a few catalase molecules. This primary effect seems to be very rare, but is efficiently enhanced by the generation of "secondary singlet oxygen" through the interaction between H2O2 and peroxynitrite at the site of inactivated catalase. Transmission of bystander signaling between pretreated and untreated tumor cells depends on the generation of secondary singlet oxygen by the pretreated cells and singlet oxygen-mediated catalase inactivation of the untreated recipient cells. This induces autoamplificatory propagation of secondary singlet oxygen generation in the population. This experimental approach allowed to quantify the efficiencies of primary and secondary singlet oxgen generation after CAP and PAM action, to dissect the system and to study the underlying chemical biology in detail. Our data confirm that CAP and PAM-derived components are merely the trigger for the activation of autoamplificatory mechanisms of tumor cells, whereas the tumor cells efficiently propagate their cell death through their own ROS/RNS signaling potential.


Assuntos
Gases em Plasma/farmacologia , Transdução de Sinais/efeitos dos fármacos , Oxigênio Singlete/metabolismo , Oxirredutases do Álcool/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Espaço Intracelular/metabolismo , Redes e Vias Metabólicas , Modelos Biológicos , NADPH Oxidase 1/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Nitritos/farmacologia
16.
Med Hypotheses ; 129: 109245, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31371071

RESUMO

Hyperhomocysteinemia (Hhcy) is a biochemical alteration with plasma levels of homocysteine higher than 15 µmol/L, associated with atherosclerosis, and with vascular thrombosis by disrupting endothelial cells. Homocysteine is a sulfur-containing amino acid derived from methionine which is an essential amino acid. Excess homocysteine produced in the body is expelled out by liver and kidney from the systemic circulation. Hhcy is caused by the excess deficiencies of the vitamins like pyridoxine (B6), folic acid (B9), or cyanocobalamin (B12). High protein consumers are usually at risk for hyperhomocysteinemia because of low plasma B12 levels. It is approximated that mild Hhcy occurs in 5-7% of the general population and 40% in patients with vascular disease. Patients with heart failure, impaired renal function, and diabetes should be screened since the prevalence of Hhcy in these patients appears to be quite high. In this article, we hypothesise that citicoline is a novel drug for the management of Hhcy. Furthermore, the side effects of citicoline are also minimal and self-limiting. If this strategy is validated, citicoline will be the cost-effective way to be administered for Hhcy. Many evidences are available which suggest that ignoring homocysteine levels in patients with the vascular disease would be unwise. Thus, there is an urgent need for health care providers to develop effective preventions and interventions program (folic acid, Vitamin B6 and Vitamin B12 supplementation as well as lifestyle change) to reduce this disorder.


Assuntos
Ácido Fólico/uso terapêutico , Hiper-Homocisteinemia/terapia , Vitamina B 12/uso terapêutico , Vitamina B 6/uso terapêutico , Oxirredutases do Álcool/metabolismo , Animais , Colina/uso terapêutico , Citidina Difosfato Colina/uso terapêutico , Suplementos Nutricionais , Células Endoteliais , Homocisteína/metabolismo , Humanos , Hidrólise , Hiper-Homocisteinemia/complicações , Rim/metabolismo , Rim/fisiopatologia , Estilo de Vida , Fígado/metabolismo , Metionina/metabolismo , Modelos Teóricos
17.
Analyst ; 144(18): 5624-5636, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31432883

RESUMO

Challenges remain in the facile, rapid and sensitive detection of substances at ultralow levels. In the current study, visual sensors of hydrogen peroxide (H2O2) and choline are developed via the integration of an ultrafine fibrous substrate and self-propagating and aggregation-induced emission (AIE) probes. Self-immolative probes (SIPs) composed of phenylboronic acid triggers and choline units are grafted on electrospun polyethylene terephthalate (PET) fibers, followed by electrostatic adsorption of tetraphenylethene derivatives (TPE-SO3) to obtain fluorescent PET-Ch/TPE fibers. Choline oxidase (ChOX) is immobilized on polystyrene-co-maleic anhydride (PSMA) fibers to obtain PSMA-ChOX, followed by assembly into PET-Ch/TPE@PSMA-ChOX composite mats. The presence of H2O2 initiates the cleavage of phenylboronic acid triggers in SIPs to release choline and choline/TPE complexes from PET-Ch/TPE fibers. The released choline is oxidized by PSMA-ChOX fibers to generate H2O2 that then activates a cascade of self-propagating reactions until the release of all choline/TPE complexes, leading to the alleviation of AIE effect and gradual fluorescence fading of fibrous mats. Thus, the hydrogen peroxide and choline concentrations can be read out from the fluorescence fading time of fibrous mats with a detection limit of 0.5 µM H2O2 within 30 min, providing potential self-test devices for a real-time, naked-eye and sensitive detection of bioactive substances.


Assuntos
Técnicas Biossensoriais/métodos , Colina/análise , Colina/química , Corantes Fluorescentes/química , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/química , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Limite de Detecção , Anidridos Maleicos/química , Modelos Moleculares , Polietilenotereftalatos/química , Poliestirenos/química , Conformação Proteica
18.
Int J Biol Macromol ; 138: 781-790, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31351953

RESUMO

To exploit robust biocatalysts for chiral 1-(2-halophenyl)ethanols synthesis, an ortho-haloacetophenones-specific carbonyl reductase (BaSDR1) gene from Bacillus aryabhattai was cloned and expressed in Escherichia coli. The impressive properties regarding BaSDR1 application include preference for NADH as coenzyme, noticeable tolerance against high cosubstrate concentration, and remarkable catalytic performance over a broad pH range from 5.0 to 10.0. The optimal temperature was 35 °C, with a half-life of 3.1 h at 35 °C and 0.75 h at 45 °C, respectively. Notably, BaSDR1 displayed excellent catalytic performance toward various ortho-haloacetophenones, providing chiral 1-(2-halophenyl)ethanols with 99% ee for all the substrates tested. Most importantly, the docking results indicated that the enzyme-substrate interactions and the steric hindrance of halogen atoms act in a push-pull manner in regulating enzyme catalytic ability. These results provide valuable clues for the structure-function relationships of BaSDR1 and the role of halogen groups in catalytic performance, and offer important reference for protein engineering and mining of functional compounds.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Bacillus/genética , Proteínas de Bactérias/genética , Catálise , Clonagem Molecular , Expressão Gênica , Concentração de Íons de Hidrogênio , Íons/química , Metais/química , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Especificidade por Substrato
19.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140255, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31349060

RESUMO

D-2-hydroxyglutaric aciduria is a neurometabolic disorder, characterized by the accumulation of D-2-hydroxyglutarate (D-2HG) in human mitochondria. Increased levels of D-2HG are detected in humans exhibiting point mutations in the genes encoding isocitrate dehydrogenase, citrate carrier, the electron transferring flavoprotein (ETF) and its downstream electron acceptor ETF-ubiquinone oxidoreductase or D-2-hydroxyglutarate dehydrogenase (hD2HGDH). However, while the pathogenicity of several amino acid replacements in the former four proteins has been studied extensively, not much is known about the effect of certain point mutations on the biochemical properties of hD2HGDH. Therefore, we recombinantly produced wild type hD2HGDH as well as two recently identified disease-related variants (hD2HGDH-I147S and -V444A) and performed their detailed biochemical characterization. We could show that hD2HGDH is a FAD dependent protein, which is able to catalyze the oxidation of D-2HG and D-lactate to α-ketoglutarate and pyruvate, respectively. The two variants were obtained as apo-proteins and were thus catalytically inactive. The addition of FAD failed to restore enzymatic activity of the variants, indicating that the cofactor binding site is compromised by the single amino acid replacements. Further analyses revealed that both variants form aggregates that are apparently unable to bind the FAD cofactor. Since, D-2-hydroxyglutaric aciduria may also result from a loss of function of either the ETF or its downstream electron acceptor ETF-ubiquinone oxidoreductase, ETF may serve as the cognate electron acceptor of reduced hD2HGDH. Here, we show that hD2HGDH directly reduces recombinant human ETF, thus establishing a metabolic link between the oxidation of D-2-hydroxyglutarate and the mitochondrial electron transport chain.


Assuntos
Oxirredutases do Álcool/química , Encefalopatias Metabólicas Congênitas/enzimologia , Mutação de Sentido Incorreto , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Substituição de Aminoácidos , Encefalopatias Metabólicas Congênitas/genética , Catálise , Flavoproteínas Transferidoras de Elétrons/química , Flavoproteínas Transferidoras de Elétrons/metabolismo , Glutaratos/química , Glutaratos/metabolismo , Humanos , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Ácido Láctico/química , Ácido Láctico/metabolismo
20.
Food Chem ; 293: 285-290, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151613

RESUMO

Exogenous adenosine triphosphate (ATP) treatment at 0, 250, 500, 750, and 1000 µM retarded cap browning in mushrooms by 0, 34, 26, 51 and 32 %, respectively, during storage at 4 °C for 18 days. Triggering signaling H2O2 accumulation arising from elevating NADPH oxidase enzyme activity during 6 days of storage at 4 °C may be pivotal for promoting shikimate dehydrogenase enzyme activity in mushrooms treated with ATP during 18 days of storage at 4 °C. Promoting melatonin accumulation (390 µg kg-1 FW vs. 160 µg kg-1 FW) in mushrooms treated with ATP during cold storage may attribute to signaling H2O2 accumulation. Higher DPPH scavenging capacity (72 % vs. 65 %) in mushrooms treated with ATP may attribute to higher phenols accumulation arising from higher phenylalanine ammonialyase/polyphenol oxidase enzymes activity concomitant with higher alternative oxidase gene expression during 18 days of storage at 4 °C.


Assuntos
Trifosfato de Adenosina/farmacologia , Agaricus/efeitos dos fármacos , Temperatura Baixa , Armazenamento de Alimentos , Reação de Maillard , Trifosfato de Adenosina/administração & dosagem , Agaricus/enzimologia , Agaricus/fisiologia , Oxirredutases do Álcool/metabolismo , Compostos de Bifenilo/química , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/metabolismo , Melatonina/metabolismo , Proteínas Mitocondriais/genética , NADPH Oxidases/metabolismo , Oxirredutases/genética , Fenóis/metabolismo , Picratos/química , Proteínas de Plantas/genética , Transdução de Sinais
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