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1.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810284

RESUMO

There is an increasing interest in polyphenols, plant secondary metabolites, in terms of fruit quality and diet, mainly due to their antioxidant effect. However, the identification of key gene enzymes and their roles in the phenylpropanoid pathway in temperate fruits species remains uncertain. Apricot (Prunus armeniaca) is a Mediterranean fruit with high diversity and fruit quality properties, being an excellent source of polyphenol compounds. For a better understanding of the phenolic pathway in these fruits, we selected a set of accessions with genetic-based differences in phenolic compounds accumulation. HPLC analysis of the main phenolic compounds and transcriptional analysis of the genes involved in key steps of the polyphenol network were carried out. Phenylalanine ammonia-lyase (PAL), dihydroflavonol-4-reductase (DFR) and flavonol synthase (FLS) were the key enzymes selected. Orthologous of the genes involved in transcription of these enzymes were identified in apricot: ParPAL1, ParPAL2, ParDFR, ParFLS1 and ParFLS2. Transcriptional data of the genes involved in those critical points and their relationships with the polyphenol compounds were analyzed. Higher expression of ParDFR and ParPAL2 has been associated with red-blushed accessions. Differences in expression between paralogues could be related to the presence of a BOXCOREDCPAL cis-acting element related to the genes involved in anthocyanin synthesis ParFLS2, ParDFR and ParPAL2.


Assuntos
Metaboloma , Polifenóis/biossíntese , Prunus/metabolismo , Transcriptoma , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polifenóis/genética , Prunus/genética
2.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669030

RESUMO

Potato tuber dormancy is critical for the post-harvest quality. Snakin/Gibberellic Acid Stimulated in Arabidopsis (GASA) family genes are involved in the plants' defense against pathogens and in growth and development, but the effect of Snakin-2 (SN2) on tuber dormancy and sprouting is largely unknown. In this study, a transgenic approach was applied to manipulate the expression level of SN2 in tubers, and it demonstrated that StSN2 significantly controlled tuber sprouting, and silencing StSN2 resulted in a release of dormancy and overexpressing tubers showed a longer dormant period than that of the control. Further analyses revealed that the decrease expression level accelerated skin cracking and water loss. Metabolite analyses revealed that StSN2 significantly down-regulated the accumulation of lignin precursors in the periderm, and the change of lignin content was documented, a finding which was consistent with the precursors' level. Subsequently, proteomics found that cinnamyl alcohol dehydrogenase (CAD), caffeic acid O-methyltransferase (COMT) and peroxidase (Prx), the key proteins for lignin synthesis, were significantly up-regulated in silencing lines, and gene expression and enzyme activity analyses also supported this effect. Interestingly, we found that StSN2 physically interacts with three peroxidases catalyzing the oxidation and polymerization of lignin. In addition, SN2 altered the hydrogen peroxide (H2O2) content and the activities of superoxide dismutase (SOD) and catalase (CAT). These results suggest that StSN2 negatively regulates lignin biosynthesis and H2O2 accumulation, and ultimately inhibits the sprouting of potato tubers.


Assuntos
Cisteína/metabolismo , Peróxido de Hidrogênio/metabolismo , Lignina/biossíntese , Proteínas de Plantas/metabolismo , Tubérculos/metabolismo , Solanum tuberosum/metabolismo , Oxirredutases do Álcool/metabolismo , Catalase/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica , Lignina/metabolismo , Peroxidase/metabolismo , Dormência de Plantas/genética , Proteínas de Plantas/genética , Caules de Planta/citologia , Caules de Planta/genética , Caules de Planta/metabolismo , Tubérculos/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ligação Proteica , Proteína O-Metiltransferase/metabolismo , Proteômica , Plântula/citologia , Plântula/genética , Plântula/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Superóxido Dismutase-1/metabolismo
3.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540582

RESUMO

Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.


Assuntos
Oxirredutases do Álcool/genética , Bacillaceae/enzimologia , Mutação , Oxirredutases do Álcool/metabolismo , Bacillaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Cinética , Metanol/metabolismo
4.
Int J Mol Sci ; 22(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562355

RESUMO

Synphilin-1 has previously been identified as an interaction partner of α-Synuclein (αSyn), a primary constituent of neurodegenerative disease-linked Lewy bodies. In this study, the repercussions of a disrupted glyoxalase system and aldose reductase function on Synphilin-1 inclusion formation characteristics and cell growth were investigated. To this end, either fluorescent dsRed-tagged or non-tagged human SNCAIP, which encodes the Synphilin-1 protein, was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast strains devoid of enzymes Glo1, Glo2, and Gre3. Presented data shows that lack of Glo2 and Gre3 activity in S. cerevisiae increases the formation of large Synphilin-1 inclusions. This correlates with enhanced oxidative stress levels and an inhibitory effect on exponential growth, which is most likely caused by deregulation of autophagic degradation capacity, due to excessive Synphilin-1 aggresome build-up. These findings illustrate the detrimental impact of increased oxidation and glycation on Synphilin-1 inclusion formation. Similarly, polar-localised inclusions were observed in wild-type S. pombe cells and strains deleted for either glo1+ or glo2+. Contrary to S. cerevisiae, however, no growth defects were observed upon expression of SNCAIP. Altogether, our findings show the relevance of yeasts, especially S. cerevisiae, as complementary models to unravel mechanisms contributing to Synphilin-1 pathology in the context of neurodegenerative diseases.


Assuntos
Aldeído Redutase/antagonistas & inibidores , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Lactoilglutationa Liase/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Glicosilação , Humanos , Corpos de Inclusão , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Oxirredução , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
5.
Toxicol Lett ; 342: 50-57, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33581289

RESUMO

Carbonyl reduction biotransformation pathway of anthracyclines (doxorubicin, daunorubicin) is a significant process, associated with drug metabolism and elimination. However, it also plays a pivotal role in anthracyclines-induced cardiotoxicity and cancer resistance. Herein, carbonyl reduction of eight anthracyclines, at in vivo relevant concentrations (20 µM), was studied in human liver cytosol, to describe the relationship between their structure and metabolism. Significant differences of intrinsic clearance between anthracyclines, ranging from 0,62-74,9 µL/min/mg were found and associated with data from in silico analyses, considering their binding in active sites of the main anthracyclines-reducing enzymes: carbonyl reductase 1 (CBR1) and aldo-keto reductase 1C3 (AKR1C3). Partial atomic charges of carbonyl oxygen atom were also determined and considered as a factor associated with reaction rate. Structural features, including presence or absence of side-chain hydroxy group, a configuration of sugar chain hydroxy group, and tetracyclic rings substitution, affecting anthracyclines susceptibility for carbonyl reduction were identified.


Assuntos
Aclarubicina/metabolismo , Citosol/metabolismo , Doxorrubicina/análogos & derivados , Hepatócitos/metabolismo , Oxirredutases/metabolismo , Aclarubicina/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Biotransformação , Doxorrubicina/química , Doxorrubicina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Conformação Proteica
6.
BMC Plant Biol ; 21(1): 83, 2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33557758

RESUMO

BACKGROUND: Cinnamyl alcohol dehydrogenase (CAD) is an important enzyme functions at the last step in lignin monomer synthesis pathway. Our previous work found that drought induced the expressions of CmCAD genes and promoted lignin biosynthesis in melon stems. RESULTS: Here we studied the effects of abscisic acid (ABA), hydrogen peroxide (H2O2) and jasmonic acid (JA) to CmCADs under drought stress. Results discovered that drought-induced ABA, H2O2 and MeJA were prevented efficiently from increasing in melon stems pretreated with fluridone (Flu, ABA inhibitor), imidazole (Imi, H2O2 scavenger) and ibuprofen (Ibu, JA inhibitor). ABA and H2O2 are involved in the positive regulations to CmCAD1, 2, 3, and 5, and JA is involved in the positive regulations to CmCAD2, 3, and 5. According to the expression profiles of lignin biosynthesis genes, ABA, H2O2 and MeJA all showed positive regulations to CmPAL2-like, CmPOD1-like, CmPOD2-like and CmLAC4-like. In addition, positive regulations were also observed with ABA to CmPAL1-like, CmC4H and CmCOMT, with H2O2 to CmPAL1-like, CmC4H, CmCCR and CmLAC17-like, and with JA to CmCCR, CmCOMT, CmLAC11-like and CmLAC17-like. As expected, the signal molecules positively regulated CAD activity and lignin biosynthesis under drought stress. Promoter::GUS assays not only further confirmed the regulations of the signal molecules to CmCAD1~3, but also revealed the important role of CmCAD3 in lignin synthesis due to the strongest staining of CmCAD3 promoter::GUS. CONCLUSIONS: CmCADs but CmCAD4 are positively regulated by ABA, H2O2 and JA under drought stress and participate in lignin synthesis.


Assuntos
Cucumis melo/genética , Cucumis melo/metabolismo , Secas , Genes de Plantas , Lignina/biossíntese , Lignina/genética , Estresse Fisiológico/genética , Ácido Abscísico/metabolismo , Oxirredutases do Álcool/metabolismo , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Oxilipinas/metabolismo , Folhas de Planta/metabolismo
7.
Chemistry ; 27(20): 6283-6294, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33475219

RESUMO

Enzyme stereoselectivity control is still a major challenge. To gain insight into the molecular basis of enzyme stereo-recognition and expand the source of antiPrelog carbonyl reductase toward ß-ketoesters, rational enzyme design aiming at stereoselectivity inversion was performed. The designed variant Q139G switched the enzyme stereoselectivity toward ß-ketoesters from Prelog to antiPrelog, providing corresponding alcohols in high enantiomeric purity (89.1-99.1 % ee). More importantly, the well-known trade-off between stereoselectivity and activity was not found. Q139G exhibited higher catalytic activity than the wildtype enzyme, the enhancement of the catalytic efficiency (kcat /Km ) varied from 1.1- to 27.1-fold. Interestingly, the mutant Q139G did not lead to reversed stereoselectivity toward aromatic ketones. Analysis of enzyme-substrate complexes showed that the structural flexibility of ß-ketoesters and a newly formed cave together facilitated the formation of the antiPrelog-preferred conformation. In contrast, the relatively large and rigid structure of the aromatic ketones prevents them from forming the antiPrelog-preferred conformation.


Assuntos
Oxirredutases do Álcool , Álcoois , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Cetonas , Estereoisomerismo , Especificidade por Substrato
8.
Biochim Biophys Acta Gen Subj ; 1865(1): 129757, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33011339

RESUMO

BACKGROUND: Trophoblast stem (TS) cell renewal and differentiation are essential processes in placentation. Special AT-rich binding protein 1 (SATB1) is a key regulator of the TS cell stem state. In this study, we identified SATB1 downstream targets and investigated their actions. METHODS: RNA-sequencing analysis was performed in Rcho-1 TS cells expressing control or Satb1 short hairpin RNAs (shRNAs) to identify candidate SATB1 targets. Differentially regulated transcripts were validated by reverse transcription-quantitative polymerase chain reaction. The role of a target of SATB1, L-threonine 3-dehydrogenase (TDH), in the regulation of trophoblast cell development was investigated using a loss-of-function approach. RESULTS: Among the differentially regulated transcripts in SATB1 knockdown TS cells, were downregulated transcripts known to affect the TS cell stem state and upregulated transcripts characteristic of trophoblast cell differentiation. Tdh expression was exquisitely responsive to SATB1 dysregulation. Tdh expression was high in the TS cell stem state and decreased as TS cells differentiated. Treatment of Rcho-1 TS cells with a TDH inhibitor or a TDH specific shRNA inhibited cell proliferation and attenuated the expression of TS cell stem state-associated transcripts and elevated the expression of trophoblast cell differentiation-associated transcripts. TDH disruption decreased TS cell colony size, Cdx2 expression, and blastocyst outgrowth. CONCLUSIONS: Our findings indicate that the actions of SATB1 on TS cell maintenance are mediated, at least in part, through the regulation and actions of TDH. GENERAL SIGNIFICANCE: Regulatory pathways controlling TS cell dynamics dictate the functionality of the placenta, pregnancy outcomes, and postnatal health.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco/citologia , Trofoblastos/citologia , Animais , Linhagem Celular , Autorrenovação Celular , Ratos Sprague-Dawley , Células-Tronco/metabolismo , Trofoblastos/metabolismo
9.
Int J Biol Macromol ; 168: 412-427, 2021 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-33316337

RESUMO

Alcohol dehydrogenases/reductases catalyze enantioselective syntheses of versatile chiral compounds relying on direct hydride transfer from cofactor to substrates, or to an intermediate and then to substrates. Since most of the substrates catalyzed by alcohol dehydrogenases/reductases are insoluble in aqueous solutions, increasing interest has been turning to organic-aqueous systems. However, alcohol dehydrogenases/reductases are normally instable in organic solvents, leading to the unsatisfied enantioselective synthesis efficiency. The behaviors of these enzymes in organic solvents at an atomic level are unclear, thus it is of great importance to understand its structure-based mechanisms in organic-aqueous systems to improve their relative stability. Here, we summarized the accessible structures of alcohol dehydrogenases/reductases in Protein Data Bank crystallized in organic-aqueous systems, and compared the structures of alcohol dehydrogenases/reductases which have different tolerance towards organic solvents. By understanding the catalytic behaviors and mechanisms of these enzymes in organic-aqueous systems, the efficient enantioselective syntheses mediated by alcohol dehydrogenases/reductases and further challenges are also discussed through solvent engineering and enzyme-immobilization in the last decade.


Assuntos
Oxirredutases do Álcool/metabolismo , Quitosana/química , Corantes de Rosanilina/química , Poluentes da Água/química , Oxirredutases do Álcool/química , Teoria da Densidade Funcional , Concentração de Íons de Hidrogênio , Imidazóis/química , Fenômenos Magnéticos , Estruturas Metalorgânicas/química , Modelos Moleculares , Conformação Molecular , Solventes/química , Purificação da Água
10.
Food Chem ; 345: 128809, 2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-33338834

RESUMO

Herein, a new double-enzymes-modulated fluorescent assay based on the quenching of upconversion nanoparticles (UCNPs) by Fe3+ was constructed for sensitive determination of OPs. OPs can inhibit the activity of acetylcholinesterase to reduce the production of choline and further lead to the lack of H2O2 in the presence of choline oxidase. Therefore, Fe2+ cannot be converted into Fe3+, resulting in "turn-on" fluorescence of UCNPs. Under optimal conditions, an excellent linear correlation between the inhibition efficiency and the logarithm of the chlorpyrifos concentration was achieved with a detection limit (LOD) of 6.7 ng/mL in the range of 20-2000 ng/mL. The recovery for chlorpyrifos in apples and cucumbers was 89.5-97.1%. The results were consistent with those obtained by GC-MS. Overall, the integration of UCNPs into the double-enzymes-mediated Fe3+/Fe2+ conversion endows this method with desirable rapidity, sensitivity, selectivity, stability, operational simplicity, and strong anti-interference capability, holding great potential in the application of food safety.


Assuntos
Acetilcolinesterase/metabolismo , Oxirredutases do Álcool/metabolismo , Técnicas Biossensoriais/métodos , Clorpirifos/análise , Ferro/química , Limite de Detecção , Nanopartículas/química , Praguicidas/análise , Espectrometria de Fluorescência
11.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118864, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32979423

RESUMO

Lanthanides are relative newcomers to the field of cell biology of metals; their specific incorporation into enzymes was only demonstrated in 2011, with the isolation of a bacterial lanthanide- and pyrroloquinoline quinone-dependent methanol dehydrogenase. Since that discovery, the efforts of many investigators have revealed that lanthanide utilization is widespread in environmentally important bacteria, and parallel efforts have focused on elucidating the molecular details involved in selective recognition and utilization of these metals. In this review, we discuss the particular chemical challenges and advantages associated with biology's use of lanthanides, as well as the currently known lanthano-enzymes and -proteins (the lanthanome). We also review the emerging understanding of the coordination chemistry and biology of lanthanide acquisition, trafficking, and regulatory pathways. These studies have revealed significant parallels with pathways for utilization of other metals in biology. Finally, we discuss some of the many unresolved questions in this burgeoning field and their potentially far-reaching applications.


Assuntos
Oxirredutases do Álcool/genética , Bactérias/genética , Elementos da Série dos Lantanídeos/metabolismo , Transporte Proteico/genética , Oxirredutases do Álcool/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Elementos da Série dos Lantanídeos/química , Metais/metabolismo
12.
Biochim Biophys Acta Mol Basis Dis ; 1867(1): 165981, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33002578

RESUMO

The Primary Hyperoxalurias (PH) are rare disorders of metabolism leading to excessive endogenous synthesis of oxalate and recurring calcium oxalate kidney stones. Alanine glyoxylate aminotransferase (AGT), deficient in PH type 1, is a key enzyme in limiting glyoxylate oxidation to oxalate. The affinity of AGT for its co-substrate, alanine, is low suggesting that its metabolic activity could be sub-optimal in vivo. To test this hypothesis, we examined the effect of L-alanine supplementation on oxalate synthesis in cell culture and in mouse models of Primary Hyperoxaluria Type 1 (Agxt KO), Type 2 (Grhpr KO) and in wild-type mice. Our results demonstrated that increasing L-alanine in cells decreased synthesis of oxalate and increased viability of cells expressing GO and AGT when incubated with glycolate. In both wild type and Grhpr KO male and female mice, supplementation with 10% dietary L-alanine significantly decreased urinary oxalate excretion ~30% compared to baseline levels. This study demonstrates that increasing the availability of L-alanine can increase the metabolic efficiency of AGT and reduce oxalate synthesis.


Assuntos
Alanina/farmacologia , Hiperoxalúria Primária/metabolismo , Oxalatos/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Células CHO , Cricetulus , Hiperoxalúria Primária/genética , Hiperoxalúria Primária/patologia , Camundongos , Camundongos Knockout , Transaminases/genética , Transaminases/metabolismo
13.
J Med Chem ; 63(24): 15308-15332, 2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33307693

RESUMO

Tuberculosis (TB) remains one of the deadliest infectious diseases and begs the scientific community to up the ante for research and exploration of completely novel therapeutic avenues. Chemical biology-inspired design of tunable chemical tools has aided in clinical diagnosis, facilitated discovery of therapeutics, and begun to enable investigation of virulence mechanisms at the host-pathogen interface of Mycobacterium tuberculosis. This Perspective highlights chemical tools specific to mycobacterial proteins and the cell lipid envelope that have furnished rapid and selective diagnostic strategies and provided unprecedented insights into the function of the mycobacterial proteome and lipidome. We discuss chemical tools that have enabled elucidating otherwise intractable biological processes by leveraging the unique lipid and metabolite repertoire of mycobacterial species. Some of these probes represent exciting starting points with the potential to illuminate poorly understood aspects of mycobacterial pathogenesis, particularly the host membrane-pathogen interactions.


Assuntos
Mycobacterium tuberculosis/metabolismo , Tuberculose/diagnóstico , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Corantes Fluorescentes/química , Humanos , Lipídeos/química , Sondas Moleculares/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Peptidoglicano/química , Sulfatases/química , Sulfatases/metabolismo , Trealose/química , Tuberculose/microbiologia , Virulência/genética
14.
Planta ; 253(1): 3, 2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-33346890

RESUMO

MAIN CONCLUSION: Eucalyptus camaldulensis EcDQD/SDH2 and 3 combine gallate formation, dehydroquinate dehydratase, and shikimate dehydrogenase activities. They are candidates for providing the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B. The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a "classical" bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.


Assuntos
Oxirredutases do Álcool , Eucalyptus , Ácido Gálico , Oxirredutases do Álcool/metabolismo , Alumínio/toxicidade , Vias Biossintéticas/fisiologia , Eucalyptus/efeitos dos fármacos , Eucalyptus/enzimologia , Eucalyptus/genética , Ácido Gálico/metabolismo , Hidroliases/metabolismo
15.
Int J Mol Sci ; 21(24)2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333742

RESUMO

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His6-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.


Assuntos
/virologia , /enzimologia , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , /genética , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Especificidade por Substrato
16.
PLoS One ; 15(12): e0244030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332435

RESUMO

Abiotic stresses especially salinity, drought and high temperature result in considerable reduction of crop productivity. In this study, we identified AT4G18280 annotated as a glycine-rich cell wall protein-like (hereafter refer to as GRPL1) protein as a potential multistress-responsive gene. Analysis of public transcriptome data and GUS assay of pGRPL1::GUS showed a strong induction of GRPL1 under drought, salinity and heat stresses. Transgenic plants overexpressing GRPL1-3HA showed significantly higher germination, root elongation and survival rate under salt stress. Moreover, the 35S::GRPL1-3HA transgenic lines also showed higher survival rates under drought and heat stresses. GRPL1 showed similar expression patterns with Abscisic acid (ABA)-pathway genes under different growth and stress conditions, suggesting a possibility that GRPL1 might act in the ABA pathway that is further supported by the inability of ABA-deficient mutant (aba2-1) to induce GRPL1 under drought stress. Taken together, our data presents GRPL1 as a potential multi-stress responsive gene working downstream of ABA.


Assuntos
Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico , Estresse Salino , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secas , Germinação/genética , Transcriptoma
17.
Nat Commun ; 11(1): 4292, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855421

RESUMO

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L-1 h-1, a titer of 275 g L-1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.


Assuntos
Bioquímica/métodos , Butanóis/metabolismo , Enzimas/metabolismo , Acetolactato Sintase/química , Acetolactato Sintase/metabolismo , Trifosfato de Adenosina , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Bioquímica/instrumentação , Reatores Biológicos , Sistema Livre de Células , Evolução Molecular Direcionada , Enzimas/química , Enzimas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Temperatura , Termodinâmica
18.
PLoS One ; 15(5): e0232090, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357153

RESUMO

Dihydroflavonol 4-reductase (DFR), a key enzyme involved in the biosynthesis of anthocyanins, has been cloned from various species. However, little research has been conducted on this enzyme in ferns, which occupy a unique evolutionary position. In this study, we isolated two novel DFR genes from the fern Dryopteris erythrosora. In vitro enzymatic analysis revealed that DeDFR1 and DeDFR2 enzymes can catalyze dihydrokaempferol and dihydroquercetin but cannot catalyze dihydromyricetin. Amino acid sequence analysis showed that DeDFR1 and DeDFR2 have an arginine at the same substrate-specificity-determining site as that in the ferns Salvinia cucullata and Azolla filiculoides. Thus, we speculate that the Arg-type DFR is a new DFR functional type. To further verify the substrate preferences of the Arg-type DFR, an amino acid substitution assay was conducted. When N133 was mutated to R133, Arabidopsis DFR protein completely lost its catalytic activity for dihydromyricetin, as observed for DeDFR1 and DeDFR2. Additionally, heterologous expression of DeDFR2 in the Arabidopsis tt3-1 mutant resulted in increasing anthocyanin accumulation. In summary, DeDFR1 and DeDFR2 are considered to be a new type of DFR with unique structures and functions. The discovery of the Arg-type DFR provides new insights into the anthocyanin biosynthesis pathway in ferns.


Assuntos
Oxirredutases do Álcool/genética , Antocianinas/biossíntese , Dryopteris/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Catálise , Dryopteris/enzimologia , Dryopteris/metabolismo , Mutagênese Sítio-Dirigida , Filogenia , Alinhamento de Sequência , Especificidade por Substrato
19.
Appl Environ Microbiol ; 86(13)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32385078

RESUMO

Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4 Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain.IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.


Assuntos
Oxirredutases do Álcool/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/genética , Clostridium acetobutylicum/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Clostridium acetobutylicum/enzimologia , Edição de Genes
20.
J Med Chem ; 63(10): 5367-5386, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32342688

RESUMO

In search of novel drugs against tuberculosis, we previously discovered and profiled a novel hydantoin-based family that demonstrated highly promising in vitro potency against Mycobacterium. tuberculosis. The compounds were found to be noncovalent inhibitors of DprE1, a subunit of decaprenylphosphoryl-ß-d-ribose-2'-epimerase. This protein, localized in the periplasmic space of the mycobacterial cell wall, was shown to be an essential and vulnerable antimycobacterial drug target. Here, we report the further SAR exploration of this chemical family through more than 80 new analogues. Among these, the most active representatives combined submicromolar cellular potency and nanomolar target affinity with balanced physicochemical properties and low human cytotoxicity. Moreover, we demonstrate in vivo activity in an acute Mtb infection model and provide further proof of DprE1 being the target of the hydantoins. Overall, the hydantoin family of DprE1 inhibitors represents a promising noncovalent lead series for the discovery of novel antituberculosis agents.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Hidantoínas/química , Hidantoínas/farmacologia , Oxirredutases do Álcool/metabolismo , Animais , Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Feminino , Células Hep G2 , Humanos , Hidantoínas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Tuberculose/tratamento farmacológico , Tuberculose/metabolismo
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