RESUMO
Oxetanocin-A is an antitumor, antiviral, and antibacterial nucleoside. It is biosynthesized via the oxidative ring contraction of a purine nucleoside co-opted from primary metabolism. This reaction is catalyzed by a B12-dependent radical S-adenosyl-l-methionine (SAM) enzyme, OxsB, and a phosphohydrolase, OxsA. Previous experiments showed that the product of the OxsB/OxsA-catalyzed reaction is an oxetane aldehyde produced alongside an uncharacterized byproduct. Experiments reported herein reveal that OxsB/OxsA complex formation is crucial for the ring contraction reaction and that reduction of the aldehyde intermediate is catalyzed by a nonspecific dehydrogenase from the general cellular pool. In addition, the byproduct is identified as a 1,3-thiazinane adduct between the aldehyde and l-homocysteine. While homocysteine was never included in the OxsB/OxsA assays, the data suggest that it can be generated from SAM via S-adenosyl-l-homocysteine (SAH). Further study revealed that conversion of SAM to SAH is facilitated by OxsB; however, the subsequent conversion of SAH to homocysteine is due to protein contaminants that co-purify with OxsA. Nevertheless, the observed demethylation of SAM to SAH suggests possible methyltransferase activity of OxsB, and substrate methylation was indeed detected in the OxsB-catalyzed reaction. This work is significant because it not only completes the description of the oxetanocin-A biosynthetic pathway but also suggests that OxsB may be capable of methyltransferase activity.
Assuntos
Adenina/análogos & derivados , S-Adenosilmetionina/química , Adenina/biossíntese , Adenina/metabolismo , Biocatálise , Catálise , Desmetilação , Metilação , Estresse Oxidativo , Monoéster Fosfórico Hidrolases/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMO
Genome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. Despite the dominance of Saccharomyces cerevisiae as a model eukaryote, an inducible genome-wide CRISPRi library in yeast has not yet been presented. Here, we present a genome-wide, inducible CRISPRi library, based on spacer design rules optimized for S. cerevisiae. We have validated this library for genome-wide interrogation of gene function across a variety of applications, including accurate discovery of haploinsufficient genes and identification of enzymatic and regulatory genes involved in adenine and arginine biosynthesis. The comprehensive nature of the library also revealed refined spacer design parameters for transcriptional repression, including location, nucleosome occupancy and nucleotide features. CRISPRi screens using this library can identify genes and pathways with high precision and a low false discovery rate across a variety of experimental conditions, enabling rapid and reliable assessment of genetic function and interactions in S. cerevisiae.
Assuntos
Sistemas CRISPR-Cas , Biblioteca Gênica , Saccharomyces cerevisiae/genética , Adenina/biossíntese , Arginina/biossíntese , Genes Fúngicos , PlasmídeosRESUMO
The use of Paecilomyces tenuipes (P. tenuipes), a Chinese medicinal fungus in scientific research, is limited due to its low adenosine content. To improve adenosine production, the present study investigated the gene network of adenosine biosynthesis in P. tenuipes via transcriptome analysis. Mycelia of P. tenuipes cultured for 24 h (PT24), 102 h (PT102) and 196 h (PT192) were subjected to RNA sequencing. In total, 13,353 unigenes were obtained. Based on sequence similarity, 8,099 unigenes were annotated with known proteins. Of these 8,099 unigenes, 5,123 had functions assigned based on Gene Ontology terms while 4,158 were annotated based on the Eukaryotic Orthologous Groups database. Moreover, 1,272 unigenes were mapped to 281 Kyoto Encyclopedia of Genes and Genomes pathways. In addition, the differential gene expression of the three libraries was also performed. A total of 601, 1,658 and 628 differentially expressed genes (DEGs) were detected in PT24 vs. PT102, PT24 vs. PT192 and PT102 vs. PT192 groups, respectively. Reverse transcriptionquantitative PCR was performed to analyze the expression levels of 14 DEGs putatively associated with adenosine biosynthesis in P. tenuipes. The results showed that two DEGs were closely associated with adenosine accumulation of P. tenuipes. The present study not only provides an improved understanding of the genetic information of P. tenuipes but also the findings can be used to aid research into P. tenuipes.
Assuntos
Adenina/biossíntese , Vias Biossintéticas , Cordyceps/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Cordyceps/genética , Cordyceps/metabolismo , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Sequência de RNA , Sequenciamento do ExomaRESUMO
Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.
Assuntos
Adenina/biossíntese , Vias Biossintéticas/genética , Carboxiliases/genética , Cromossomos Artificiais Humanos/química , Peptídeo Sintases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sistemas CRISPR-Cas , Carboxiliases/metabolismo , Cromossomos Artificiais Humanos/metabolismo , Teste de Complementação Genética , Engenharia Genética/métodos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Peptídeo Sintases/metabolismo , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Covering: 2011 to 2018 This highlight summarizes the investigation of cobalamin (Cbl)- and radical S-adenosyl-l-methionine (SAM)-dependent enzymes found in natural product biosynthesis to date and suggests some possibilities for the future. Though some mechanistic aspects are apparently shared, the overall diversity of this family's functions and abilities is significant and may be tailored to the specific substrate and/or reaction being catalyzed. A little over a year ago, the first crystal structure of a Cbl- and radical SAM-dependent enzyme was solved, providing the first insight into what may be the shared scaffolding of these enzymes.
Assuntos
Produtos Biológicos/metabolismo , Enzimas/química , Enzimas/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismo , Adenina/análogos & derivados , Adenina/biossíntese , Aminobutiratos/metabolismo , Fosfomicina/biossíntese , Gentamicinas/biossíntese , Metilação , Shewanella/enzimologia , Tioestreptona/biossínteseRESUMO
AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism. Recently we found that upregulated AMPD activity is associated with ATP depletion and contractile dysfunction under the condition of pressure overloading in the heart of a rat model of type 2 diabetes mellitus (T2DM), OLETF. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates by 55% in OLETF compared to those in LETO, a non-diabetic control. In contrast, the mRNA levels of AMPD3 in the myocardium were similar in OLETF and LETO. AMPD3 was comparably ubiquitinated in OLETF and LETO, and its degradation ex vivo was more sensitive to MG-132, a proteasome inhibitor, in OLETF than in LETO. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3'UTR of AMPD3 mRNA. AMPD3 protein level was significantly increased by a miR-301b inhibitor and was decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay confirmed binding of miR-301b to the 3'UTR of AMPD3 mRNA. Transfection of neonatal rat cardiomyocytes with a miR-301b inhibitor increased 90-kDa AMPD3 and reduced ATP level. The results indicate that translational regulation by miR-301b mediates upregulated expression of cardiac AMPD3 protein in OLETF, which potentially reduces the adenine nucleotide pool at the time of increased work load. The miR-301b-AMPD3 axis may be a novel therapeutic target for intervening enegy metabolism in diabetic hearts.
Assuntos
AMP Desaminase/genética , Diabetes Mellitus Tipo 2/genética , MicroRNAs/genética , Miocárdio/metabolismo , Adenina/biossíntese , Trifosfato de Adenosina/genética , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Contração Miocárdica/genética , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RatosRESUMO
Covering: up to the end of 2017 C-C bond formations are frequently the key steps in cofactor and natural product biosynthesis. Historically, C-C bond formations were thought to proceed by two electron mechanisms, represented by Claisen condensation in fatty acids and polyketide biosynthesis. These types of mechanisms require activated substrates to create a nucleophile and an electrophile. More recently, increasing number of C-C bond formations catalyzed by radical SAM enzymes are being identified. These free radical mediated reactions can proceed between almost any sp3 and sp2 carbon centers, allowing introduction of C-C bonds at unconventional positions in metabolites. Therefore, free radical mediated C-C bond formations are frequently found in the construction of structurally unique and complex metabolites. This review discusses our current understanding of the functions and mechanisms of C-C bond forming radical SAM enzymes and highlights their important roles in the biosynthesis of structurally complex, naturally occurring organic molecules. Mechanistic consideration of C-C bond formation by radical SAM enzymes identifies the significance of three key mechanistic factors: radical initiation, acceptor substrate activation and radical quenching. Understanding the functions and mechanisms of these characteristic enzymes will be important not only in promoting our understanding of radical SAM enzymes, but also for understanding natural product and cofactor biosynthesis.
Assuntos
Produtos Biológicos/química , Coenzimas/biossíntese , Enzimas/química , Enzimas/metabolismo , S-Adenosilmetionina/metabolismo , Adenina/análogos & derivados , Adenina/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/biossíntese , Produtos Biológicos/metabolismo , Carbono/química , Coenzimas/química , Endopeptidases/química , Endopeptidases/metabolismo , Hidrolases/química , Hidrolases/metabolismo , Molibdênio/química , Molibdênio/metabolismo , Tunicamicina/biossíntese , Vitamina K 2/metabolismoRESUMO
Oxetanocin A (OXT-A) is a potent antitumour, antiviral and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne Bacillus megaterium gene cluster that contains four genes: oxsA, oxsB, oxrA and oxrB. Here we show that both the oxsA and oxsB genes are required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, reveals that OXT-A is derived from a 2'-deoxyadenosine phosphate in an OxsB-catalysed ring contraction reaction initiated by hydrogen atom abstraction from C2'. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme, a family of enzymes with an estimated 7,000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes.
Assuntos
Adenina/análogos & derivados , Bacillus megaterium/enzimologia , Proteínas de Bactérias/metabolismo , Biocatálise , Coenzimas/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismo , Adenina/biossíntese , Monofosfato de Adenosina/metabolismo , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Nucleotídeos de Desoxiadenina/metabolismo , Genes Bacterianos/genética , Modelos Moleculares , Família Multigênica/genética , Conformação ProteicaRESUMO
Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives.
Assuntos
Adenina/metabolismo , Anti-Infecciosos/farmacologia , Butiratos/farmacologia , Penicillium/efeitos dos fármacos , Penicillium/metabolismo , Adenina/biossíntese , Adenosina/química , Adenosina/metabolismo , Biomassa , Cromatografia Líquida de Alta Pressão/métodos , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Hipoxantina/química , Hipoxantina/metabolismo , Testes de Sensibilidade Microbiana , Penicillium/química , Esporos Fúngicos/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacosRESUMO
HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of the Bacillus megaterium HD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A. These studies reveal a previously uncharacterized reaction for this family; OxsA catalyzes the conversion of a triphosphorylated compound into a nucleoside, releasing one molecule of inorganic phosphate at a time. Remarkably, this functionality is a result of the OxsA active site, which based on structural and kinetic analyses has been tailored to bind the small, four-membered ring of oxetanocin-A over larger substrates. Furthermore, our OxsA structures show an active site that switches from a dinuclear to a mononuclear metal center as phosphates are eliminated from substrate.
Assuntos
Adenina/análogos & derivados , Bacillus megaterium/enzimologia , Monoéster Fosfórico Hidrolases/química , Conformação Proteica , Adenina/biossíntese , Adenina/química , Ácido Aspártico/química , Ácido Aspártico/genética , Bacillus megaterium/química , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Histidina/química , Histidina/genética , Cinética , Monoéster Fosfórico Hidrolases/genética , Transdução de Sinais , Especificidade por SubstratoRESUMO
Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [PSI+ ] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [psi- ] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [PSI+ ] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read-through of the ade1-14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced-glutathione, which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid-forming human proteins TDP-43, Aß-42 and Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate-bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2-1, ade2Δ, and ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Amiloide/genética , Bioensaio/métodos , Mutação , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenina/biossíntese , Amiloide/metabolismo , Vias Biossintéticas/genética , Microscopia de FluorescênciaRESUMO
Gonad-stimulating substance (GSS) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. In breeding season (stage V), GSS stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) by ovarian follicle cells. The hormonal action of GSS is mediated through the activation of its receptor, G-proteins and adenylyl cyclase. It has been reported that GSS fails to induce 1-MeAde and cyclic AMP (cAMP) production in follicle cells of ovaries during oogenesis (stage IV). This study examined the regulatory mechanism how ovarian follicle cells acquire the potential to respond to GSS by producing 1-MeAde and cAMP. Because the failure of GSS action was due to G-proteins of follicle cells, the molecular structures of Gαs, Gαi, Gαq and Gß were identified in follicle cells of starfish Asterina pectinifera. The cDNA sequences of Gαs, Gαi, Gαq and Gß consisted of ORFs encoding 379, 354, 353 and 353 amino acids. The expression levels of Gαs were extremely low in follicle cells at stage IV, whereas the mRNA levels increased markedly in stage V. On contrary, the mRNA levels of Gαi were almost constant regardless of stage IV and V. These findings strongly suggest that de novo synthesis of Gαs-proteins is contributed to the action of GSS on follicle cells to produce 1-MeAde and cAMP.
Assuntos
Asterina/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Hormônios de Invertebrado/farmacologia , Neuropeptídeos/farmacologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Relaxina/metabolismo , Adenina/análogos & derivados , Adenina/biossíntese , Sequência de Aminoácidos , Animais , Asterina/efeitos dos fármacos , Sítios de Ligação , Western Blotting , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Hormônios de Invertebrado/metabolismo , Cinética , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The majority of Fe in Fe-replete yeast cells is located in vacuoles. These acidic organelles store Fe for use under Fe-deficient conditions and they sequester it from other parts of the cell to avoid Fe-associated toxicity. Vacuolar Fe is predominantly in the form of one or more magnetically isolated nonheme high-spin (NHHS) Fe(III) complexes with polyphosphate-related ligands. Some Fe(III) oxyhydroxide nanoparticles may also be present in these organelles, perhaps in equilibrium with the NHHS Fe(III). Little is known regarding the chemical properties of vacuolar Fe. When grown on adenine-deficient medium (A↓), ADE2Δ strains of yeast such as W303 produce a toxic intermediate in the adenine biosynthetic pathway. This intermediate is conjugated with glutathione and shuttled into the vacuole for detoxification. The iron content of A↓ W303 cells was determined by Mössbauer and EPR spectroscopies. As they transitioned from exponential growth to stationary state, A↓ cells (supplemented with 40 µM Fe(III) citrate) accumulated two major NHHS Fe(II) species as the vacuolar NHHS Fe(III) species declined. This is evidence that vacuoles in A↓ cells are more reducing than those in adenine-sufficient cells. A↓ cells suffered less oxidative stress despite the abundance of NHHS Fe(II) complexes; such species typically promote Fenton chemistry. Most Fe in cells grown for 5 days with extra yeast-nitrogen-base, amino acids and bases in minimal medium was HS Fe(III) with insignificant amounts of nanoparticles. The vacuoles of these cells might be more acidic than normal and can accommodate high concentrations of HS Fe(III) species. Glucose levels and rapamycin (affecting the TOR system) affected cellular Fe content. This study illustrates the sensitivity of cellular Fe to changes in metabolism, redox state and pH. Such effects broaden our understanding of how Fe and overall cellular metabolism are integrated.
Assuntos
Adenina/metabolismo , Compostos Férricos/metabolismo , Compostos Ferrosos/metabolismo , Ferro/metabolismo , Vacúolos/metabolismo , Adenina/administração & dosagem , Adenina/biossíntese , Benzamidas/farmacologia , Benzodioxóis/farmacologia , Meios de Cultura/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Modelos Biológicos , Ferroproteínas não Heme/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Espectroscopia de MossbauerRESUMO
IC87114 is a selective PI3Kδ inhibitor. A simple, sensitive and reliable LC-MS/MS method with rapid sample preparation was developed and validated for the determination of IC87114 in mouse plasma, bronchoalveolar lavage, and lung. Chromatographic separation was achieved using an Agilent Zorbax Eclipse XDB-C18 column (150mm×2.1mm internal diameter, 3.5µm particle size). Mass spectrometric detection was conducted by electrospray ionization in positive ion multiple reaction monitoring modes. The calibration curve was linear over a concentration range of 0.01-1000ng/mL for plasma/BAL and 0.1-250ng/mL for lung tissue. Recoveries were as high as 97.29%, 102.81% and 89.70% for plasma, BAL fluid and lung sample, respectively. The lower limit of quantification was 0.01ng/mL. Intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. Finally, the method was successfully used in a pharmacokinetic study that measured IC87114 in mouse plasma, BAL fluid and lung tissue after administration of a single 1mg/kg intratracheal dose of IC87114. The percentage change for incurred sample reanalysis (ISR) was within ±15.0% and met the acceptance criteria for ISR.
Assuntos
Adenina/análogos & derivados , Cromatografia Líquida/métodos , Pulmão/metabolismo , Quinazolinas/farmacocinética , Espectrometria de Massas em Tandem/métodos , Adenina/biossíntese , Adenina/metabolismo , Adenina/farmacocinética , Animais , Lavagem Broncoalveolar/métodos , Camundongos , Camundongos Endogâmicos C57BL , Quinazolinas/metabolismoRESUMO
Genetic and biochemical studies have recently implicated four proteins required in bacteria for the biosynthesis of the universal tRNA modified base N6-threonylcarbamoyl adenosine (t(6)A). In this work, t(6)A biosynthesis in Bacillus subtilis has been reconstituted in vitro and found to indeed require the four proteins YwlC (TsaC), YdiB (TsaE), YdiC (TsaB) and YdiE (TsaD). YwlC was found to catalyze the conversion of L-threonine, bicarbonate/CO(2) and ATP to give the intermediate L-threonylcarbamoyl-AMP (TC-AMP) and pyrophosphate as products. TC-AMP was isolated by HPLC and characterized by mass spectrometry and (1)H NMR. NMR analysis showed that TC-AMP decomposes to give AMP and a nearly equimolar mixture of L-threonine and 5-methyl-2-oxazolidinone-4-carboxylate as final products. Under physiological conditions (pH 7.5, 37 °C, 2 mM MgCl(2)), the half-life of TC-AMP was measured to be 3.5 min. Both YwlC (in the presence of pyrophosphatase) and its Escherichia coli homologue YrdC catalyze the formation of TC-AMP while producing only a small molar fraction of AMP. This suggests that CO(2) and not an activated form of bicarbonate is the true substrate for these enzymes. In the presence of pyrophosphate, both enzymes catalyze clean conversion of TC-AMP back to ATP. Purified TC-AMP is efficiently processed to t(6)A by the YdiBCE proteins in the presence of tRNA substrates. This reaction is ATP independent in vitro, despite the known ATPase activity of YdiB. The estimated rate of conversion of TC-AMP by YdiBCE to t(6)A is somewhat lower than the initial rate from L-threonine, bicarbonate and ATP, which together with the stability data, is consistent with previous studies that suggest channeling of this intermediate.
Assuntos
Adenina/análogos & derivados , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/biossíntese , Treonina/análogos & derivados , Adenina/biossíntese , Monofosfato de Adenosina/isolamento & purificação , Oxirredutases do Álcool/metabolismo , Bacillus subtilis/enzimologia , Cinética , Especificidade por Substrato , Treonina/biossíntese , Treonina/isolamento & purificaçãoRESUMO
In the laboratory, students can actively explore concepts and experience the nature of scientific research. We have devised a 5-wk laboratory project in our introductory college biology course whose aim was to improve understanding in five major concepts that are central to basic cellular, molecular biology, and genetics while teaching molecular biology techniques. The project was focused on the production of adenine in Saccharomyces cerevisiae and investigated the nature of mutant red colonies of this yeast. Students created red mutants from a wild-type strain, amplified the two genes capable of giving rise to the red phenotype, and then analyzed the nucleotide sequences. A quiz assessing student understanding in the five areas was given at the start and the end of the course. Analysis of the quiz showed significant improvement in each of the areas. These areas were taught in the laboratory and the classroom; therefore, students were surveyed to determine whether the laboratory played a role in their improved understanding of the five areas. Student survey data demonstrated that the laboratory did have an important role in their learning of the concepts. This project simulated steps in a research project and could be adapted for an advanced course in genetics.
Assuntos
Adenina/biossíntese , Biologia Celular/educação , Técnicas de Laboratório Clínico , Biologia Molecular/educação , Saccharomyces cerevisiae/metabolismo , DNA Fúngico/isolamento & purificação , Humanos , Mutação , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética , Estudantes , Raios UltravioletaRESUMO
Fruit bodies and mycelia of shiitake mushroom ( Lentinus edodes) have been shown to contain the cholesterol-reducing compound eritadenine, 2( R),3( R)-dihydroxy-4-(9-adenyl)butyric acid. In the search for a production method for eritadenine, shiitake mycelia were investigated in the present study. The mycelia were cultivated both in shake flasks and in bioreactors, to investigate the effects of pH, stirring rate, and reactor type on the production and distribution of eritadenine. Both the biomass and the culture broth were examined for their eritadenine content. In the shake flasks, the final concentration of eritadenine was 1.76 mg/L and eritadenine was equally distributed between the mycelia and the growth media. In the bioreactors, the shiitake mycelia were found to contain eritadenine in relatively low levels, whereas the majority, 90.6-98.9%, was detected in the growth media. Applying a stirring rate of 250 rpm during bioreactor cultivation resulted in the highest eritadenine concentrations: 10.23 mg/L when the pH was uncontrolled and 9.59 mg/L when the pH was controlled at 5.7. Reducing the stirring rate to 50 rpm resulted in a decreased eritadenine concentration, both at pH 5.7 (5.25 mg/L) and when pH was not controlled (5.50 mg/L). The mycelia in the shake flask cultures appeared as macroscopic aggregates, whereas mycelia cultivated in bioreactors grew more as freely dispersed filaments. This study demonstrates for the first time the extra- and intracellular distribution of eritadenine produced by shiitake mycelial culture and the influence of reactor conditions on the mycelial morphology and eritadenine concentrations.
Assuntos
Adenina/análogos & derivados , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Cogumelos Shiitake/crescimento & desenvolvimento , Cogumelos Shiitake/metabolismo , Adenina/biossíntese , Reatores Biológicos , Concentração de Íons de HidrogênioRESUMO
BACKGROUND: Consumption of farm milk in early life is associated with less asthma and allergies. OBJECTIVE: We hypothesized that genetic variation in the innate immunity receptor CD14 might modify the association between farm milk consumption and asthma and atopy. METHODS: Questionnaire data, serum IgE levels, and genotypes for 4 single nucleotide polymorphisms in CD14 were assessed in farmers' and nonfarmers' children from 2 European populations (Allergy and Endotoxin study, n = 576; Prevention of Allergy Risk factors for Sensitization in children related to Farming and Anthroposophic Lifestyle study, n = 1539). In a subsample (n = 222) CD14 gene expression was measured in peripheral blood leukocytes. The effects of farm milk and CD14 genotypes on asthma, allergies, and CD14 expression and their interactions were investigated. RESULTS: We found a significant interaction between genetic variation in CD14/-1721 and farm milk consumption. Adjusted odds ratios for the association between farm milk and asthma varied between the genotypes: AA, 0.18 (95% CI, 0.07-0.47); AG, 0.47 (95% CI, 0.26-0.86); and GG, 0.98 (95% CI, 0.46-2.08). Similar patterns were observed for symptoms of allergic rhinoconjunctivitis and pollen sensitization. CD14/-1721 also modified the association between farm milk and CD14 gene expression (adjusted geometric means ratios: AA, 1.61 (95% CI, 0.98-2.66); AG, 1.11 (95% CI, 0.71-1.72); and GG, 0.76 (95% CI, 0.39-1.48). CONCLUSION: The protective effect of farm milk consumption on allergic diseases is stronger in children carrying the A allele in CD14/-1721 than in children homozygous for the G allele. This might be mediated through farm milk-induced upregulated CD14 gene expression. CLINICAL IMPLICATIONS: Our results support the hypothesis that the inverse association between farm milk consumption and allergic diseases is mediated by CD14-activated innate immune mechanisms.
Assuntos
Hipersensibilidade Alimentar/genética , Hipersensibilidade Alimentar/imunologia , Regulação da Expressão Gênica/imunologia , Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/genética , Leite/imunologia , Polimorfismo de Nucleotídeo Único , Adenina/biossíntese , Alelos , Animais , Criança , Estudos Transversais , Indústria de Laticínios , Feminino , Genótipo , Guanina/biossíntese , Humanos , Imunidade Inata/genética , Lactente , MasculinoRESUMO
In this work, a fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032 was investigated. Nicotinamide adenine dinucleotide (NADH) production and formate dehydrogenase activity increased with formate addition from 0.5 to 2.0 g/L, respectively. However, with the formate addition of 1.5 g/L, the activities of pyruvate kinase and glucose 6-phosphate dehydrogenase reached a peak and increased by 316 and 150% relative to those of the control, respectively. In addition, intracellular production of pyruvate, aspartate, citrate and adenine were significantly enhanced by 75, 66, 32 and 78% as well. An improvement (90%) of thuringiensin production was also successfully obtained. Interestingly to point out, thuringiensin yield was closely correlative with adenine production, and the linear relationship was also observed. The results suggest that appropriate formate addition did act as a modulator and facilitate carbon flux in glycolysis and pentose phosphate pathway to synthesize adenine and thuringiensin via intracellular NADH availability.
Assuntos
Adenina/biossíntese , Adenosina/análogos & derivados , Bacillus thuringiensis/metabolismo , Formiatos/administração & dosagem , Regulação Bacteriana da Expressão Gênica/fisiologia , Transdução de Sinais/fisiologia , Adenosina/biossíntese , Bacillus thuringiensis/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/fisiologia , Transdução de Sinais/efeitos dos fármacos , Açúcares ÁcidosRESUMO
Purine nucleotides are critically important for the normal functioning of cells due to their myriad of activities. It is important for cells to maintain a balance in the pool sizes of the adenine-containing and guanine-containing nucleotides, which occurs by a combination of de novo synthesis and salvage pathways that interconvert the purine nucleotides. This review describes the mechanism for regulation of the biosynthetic genes in the yeast Saccharomyces cerevisiae and compares this mechanism with that described in several microbial species.