RESUMO
Genome homology and the presence of a putative biosynthetic gene cluster identified Streptomyces aureorectus DSM 41692 and Streptomyces virens DSM 41465 as candidate producers of the antibiotic nucleocidin 1. Indeed when these bacterial strains were cultured in a medium supplemented with fluoride (4 mM) they each produced nucleocidin 1 and the previously identified 4'-fluoro-3'-O-ß-glucosylated adenosine 2 and its sulfamylated derivative 3. In both of these cases 4'-fluoroadenosine 9 is also identified as a natural product although it has never been observed during fermentations of Streptomyces calvus, the original source of nucleocidin 1. The identity of 4'-fluoroadenosine 9 was confirmed by a total synthesis as well as by its in vitro enzymatic conversion to metabolite 2 using the glucosyl transferase enzyme, NucGT.
Assuntos
Adenosina/análogos & derivados , Antibacterianos/biossíntese , Streptomyces/metabolismo , Adenosina/biossíntese , Adenosina/química , Antibacterianos/química , Estrutura Molecular , Filogenia , Espectroscopia de Prótons por Ressonância Magnética , Streptomyces/classificaçãoRESUMO
Angustmycin A has anti-mycobacterial and cytokinin activities, and contains an intriguing structure in which an unusual sugar with C5'-C6' dehydration is linked to adenine via an N-glycosidic bond. However, the logic underlying the biosynthesis of this molecule has long remained obscure. Here, we address angustmycin A biosynthesis by the full deciphering of its pathway. We demonstrate that AgmD, C, A, E, and B function as D-allulose 6-phosphate 3-epimerase, D-allulose 6-phosphate pyrophosphokinase, adenine phosphoallulosyltransferase, phosphoribohydrolase, and phosphatase, respectively, and that these collaboratively catalyze the relay reactions to biosynthesize angustmycin C. Additionally, we provide evidence that AgmF is a noncanonical dehydratase for the final step to angustmycin A via a self-sufficient strategy for cofactor recycling. Finally, we have reconstituted the entire six-enzyme pathway in vitro and in E. coli leading to angustmycin A production. These results expand the enzymatic repertoire regarding natural product biosynthesis, and also open the way for rational and rapid discovery of other angustmycin related antibiotics.
Assuntos
Adenosina/análogos & derivados , Citocininas/biossíntese , Nucleosídeos/biossíntese , Adenosina/biossíntese , Adenosina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Citocininas/química , Desidratação , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Estrutura Molecular , Família Multigênica , Nucleosídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
A key objective in immuno-oncology is to reactivate the dormant immune system and increase tumour immunogenicity. Adenosine is an omnipresent purine that is formed in response to stress stimuli in order to restore physiological balance, mainly via anti-inflammatory, tissue-protective, and anti-nociceptive mechanisms. Adenosine overproduction occurs in all stages of tumorigenesis, from the initial inflammation/local tissue damage to the precancerous niche and the developed tumour, making the adenosinergic pathway an attractive but challenging therapeutic target. Many current efforts in immuno-oncology are focused on restoring immunosurveillance, largely by blocking adenosine-producing enzymes in the tumour microenvironment (TME) and adenosine receptors on immune cells either alone or combined with chemotherapy and/or immunotherapy. However, the effects of adenosinergic immunotherapy are not restricted to immune cells; other cells in the TME including cancer and stromal cells are also affected. Here we summarise recent advancements in the understanding of the tumour adenosinergic system and highlight the impact of current and prospective immunomodulatory therapies on other cell types within the TME, focusing on adenosine receptors in tumour cells. In addition, we evaluate the structure- and context-related limitations of targeting this pathway and highlight avenues that could possibly be exploited in future adenosinergic therapies.
Assuntos
Adenosina/imunologia , Terapia de Alvo Molecular , Neoplasias/imunologia , Receptores Purinérgicos P1/uso terapêutico , Adenosina/biossíntese , Adenosina/genética , Adenosina/uso terapêutico , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/imunologia , Humanos , Imunoterapia/tendências , Neoplasias/genética , Neoplasias/terapia , Receptores Purinérgicos P1/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Adenosylhopane is a crucial precursor of C35 hopanoids, which are believed to modulate the fluidity and permeability of bacterial cell membranes. Adenosylhopane is formed by a crosslinking reaction between diploptene and a 5'-deoxyadenosyl radical that is generated by the radical S-adenosyl-L-methionine (SAM) enzyme HpnH. We previously showed that HpnH from Streptomyces coelicolor A3(2) (ScHpnH) converts diploptene to (22R)-adenosylhopane. However, the mechanism of the stereoselective C-C bond formation was unclear. Thus, here, we performed biochemical and mutational analysis of another HpnH, from the ethanol-producing bacterium Zymomonas mobilis (ZmHpnH). Similar to ScHpnH, wild-type ZmHpnH afforded (22R)-adenosylhopane. Conserved cysteine and tyrosine residues were suggested as possible hydrogen sources to quench the putative radical reaction intermediate. A Cys106Ala mutant of ZmHpnH had one-fortieth the activity of the wild-type enzyme and yielded both (22R)- and (22S)-adenosylhopane along with some related byproducts. Radical trapping experiments with a spin-trapping agent supported the generation of a radical intermediate in the ZmHpnH-catalyzed reaction. We propose that the thiol of Cys106 stereoselectively reduces the radical intermediate generated at the C22 position by the addition of the 5'-deoxadenosyl radical to diploptene, to complete the reaction.
Assuntos
Adenosina/análogos & derivados , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Adenosina/biossíntese , Adenosina/genética , Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Catálise , Cisteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Triterpenos/química , Zymomonas/metabolismoRESUMO
The nucleoside antibiotic angustmycin, produced by some Streptomyces strains, is composed of adenine and C6 sugar and shows antibiotic and antitumor activities. In this study, we propose a biosynthetic pathway for angustmycin using a heterologous expression experiment coupled with in silico analysis of the angustmycin biosynthetic gene (agm) cluster. The biochemical characterization of Agm6 demonstrated its role in angustmycin biosynthesis as an unprecedented dehydratase.
Assuntos
Adenosina/biossíntese , Antibacterianos/biossíntese , Hidroliases/genética , Hidroliases/metabolismo , Família Multigênica/genética , Adenosina/genética , Simulação por Computador , Streptomyces/efeitos dos fármacosRESUMO
BACKGROUND: Staphylococcus aureus is a common human pathogen capable of causing diverse illnesses with possible recurrent infections. Although recent studies have highlighted the role of cellular immunity in recurrent infections, the mechanism by which S. aureus evades host responses remains largely unexplored. METHODS: This study utilizes in vitro and in vivo infection experiments to investigate difference of pro-inflammatory responses and subsequent adaptive immune responses between adsA mutant and WT S. aureus strain infection. FINDINGS: We demonstrated that adenosine synthase A (AdsA), a potent S. aureus virulence factor, can alter Th17 responses by interfering with NLRP3 inflammasome-mediated IL-1ß production. Specifically, S. aureus virulence factor AdsA dampens Th1/Th17 immunity by limiting the release of IL-1ß and other Th polarizing cytokines. In particular, AdsA obstructs the release of IL-1ß via the adenosine/A2aR/NLRP3 axis. Using a murine infection model, pharmacological inhibition of A2a receptor enhanced S. aureus-specific Th17 responses, whereas inhibition of NLRP3 and caspase-1 downregulated these responses. Our results showed that AdsA contributes to recurrent S. aureus infection by restraining protective Th1/Th17 responses. INTERPRETATION: Our study provides important mechanistic insights for therapeutic and vaccination strategies against S. aureus infections. FUNDING: This work was supported by grants from Shenzhen Peacock project (KQTD2015033-117210153), and Guangdong Science and Technology Department (2020B1212030004) to J.H. and China Postdoctoral Science Foundation (2019M663167) to BZZ. We also thank the L & T Charitable Foundation, the Guangdong Science and Technology Department (2020B1212030004), and the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2019BT02Y198) for their support.
Assuntos
Proteínas de Bactérias/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/enzimologia , Fatores de Virulência/imunologia , Adenosina/biossíntese , Animais , Células Cultivadas , Feminino , Humanos , Evasão da Resposta Imune , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptor A2A de Adenosina/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade , Células THP-1 , Células Th17/imunologiaRESUMO
N6-methyladenosine (m6A) is one of the most widespread and highly conserved chemical modifications in cellular RNAs of eukaryotic genomes. Owing to the development of high-throughput m6A sequencing, the functions and mechanisms of m6A modification in development and diseases have been revealed. Recent studies have shown that RNA m6A methylation plays a critical role in skeletal muscle development, which regulates myoblast proliferation and differentiation, and muscle regeneration. Exploration of the functions of m6A modification and its regulators provides a deeper understanding of the regulatory mechanisms underlying skeletal muscle development. In the present review, we aim to summarize recent breakthroughs concerning the global landscape of m6A modification in mammals and examine the biological functions and mechanisms of enzymes regulating m6A RNA methylation. We describe the interplay between m6A and other epigenetic modifications and highlight the regulatory roles of m6A in development, especially that of skeletal muscle. m6A and its regulators are expected to be targets for the treatment of human muscle-related diseases and novel epigenetic markers for animal breeding in meat production.
Assuntos
Adenosina/análogos & derivados , Epigênese Genética/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Doenças Musculares/genética , RNA/genética , Adenosina/biossíntese , Adenosina/genética , Animais , Diferenciação Celular , Humanos , Metilação , Músculo Esquelético/patologia , Doenças Musculares/metabolismo , Doenças Musculares/patologia , RNA/metabolismoRESUMO
Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) ß-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.
Assuntos
Adenosina/análogos & derivados , Antibacterianos/biossíntese , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Mutação , Streptomyces/genética , Adenosina/biossíntese , Adenosina/química , Substituição de Aminoácidos , Antibacterianos/química , Antifúngicos/química , Antifúngicos/metabolismo , Antimaláricos/química , Antimaláricos/metabolismo , Antiprotozoários/química , Antiprotozoários/metabolismo , Antivirais/química , Antivirais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA/química , DNA/genética , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Teoria da Densidade Funcional , Regulação Bacteriana da Expressão Gênica , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Streptomyces/enzimologiaRESUMO
The tRNA modification N6-threonylcarbamoyladenosine (t6A) is universally conserved in all organisms. In bacteria, the biosynthesis of t6A requires four proteins (TsaBCDE) that catalyze the formation of t6A via the unstable intermediate l-threonylcarbamoyl-adenylate (TC-AMP). While the formation and stability of this intermediate has been studied in detail, the mechanism of its transfer to A37 in tRNA is poorly understood. To investigate this step, the structure of the TsaBD heterodimer from Escherichia coli has been solved bound to a stable phosphonate isosteric mimic of TC-AMP. The phosphonate inhibits t6A synthesis in vitro with an IC50 value of 1.3 µM in the presence of millimolar ATP and L-threonine. The inhibitor binds to TsaBD by coordination to the active site Zn atom via an oxygen atom from both the phosphonate and the carboxylate moieties. The bound conformation of the inhibitor suggests that the catalysis exploits a putative oxyanion hole created by a conserved active site loop of TsaD and that the metal essentially serves as a binding scaffold for the intermediate. The phosphonate bound crystal structure should be useful for the rational design of potent, drug-like small molecule inhibitors as mechanistic probes or potentially novel antibiotics.
Assuntos
Adenosina/análogos & derivados , Proteínas de Escherichia coli/química , RNA de Transferência/metabolismo , Adenosina/biossíntese , Adenosina/química , Domínio Catalítico , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Organofosfonatos/química , Organofosfonatos/farmacologia , Multimerização Proteica , RNA de Transferência/químicaRESUMO
AIMS: N6-Methyladenosine (m6A) is the most frequent posttranscriptional modification and plays important roles in tumorigenesis and metastasis. The roles of fat mass and obesity-associated (FTO) in metabolic diseases have been widely explored. However, the molecular mechanisms and physiological functions of FTO in prostate cancer remain largely unknown. This study aimed to explore the exact functions of FTO in the progression of prostate cancer metastasis. MAIN METHODS: Dot blot and m6A RNA methylation quantification assays were performed to determine m6A levels. The protein and mRNA expression levels were detected using immunoblot (IB) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses. Cell invasion and migration abilities were measured using transwell and wound healing assays. Bioinformatics was used to measure the expression level of FTO and possible correlation between FTO levels and advanced tumor stage. Immunofluorescence (IF) was performed to measure the cellular localization of FTO. KEY FINDINGS: FTO was downregulated in prostate cancer tissues and cell lines, and the m6A content was increased. Importantly, patients with lower FTO expression had advanced tumor stage and higher Gleason scores. Gain- and loss-of-function assays revealed that FTO inhibits prostate cancer cell invasion and migration in vitro. Moreover, we confirmed that FTO can decrease the total m6A level. SIGNIFICANCE: The present study revealed that the FTO m6A demethylase inhibits prostate cancer cell invasion and migration by regulating total m6A levels.
Assuntos
Adenosina/análogos & derivados , Dioxigenase FTO Dependente de alfa-Cetoglutarato/biossíntese , Biomarcadores Tumorais/biossíntese , Movimento Celular/fisiologia , Neoplasias da Próstata/metabolismo , Adenosina/antagonistas & inibidores , Adenosina/biossíntese , Idoso , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologiaRESUMO
Adenosine and uric acid (UA) play a pivotal role in lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). In the present experiments, we measured adenosine synthesis from nicotinamide adenine dinucleotide (NAD+) in membranes prepared from wild type (WT) and CD38 knockout (CD38KO) mouse lungs, from cultured airway smooth muscle and epithelial cells, and in bronchoalveolar lavage fluid after airway challenge with epidemiologically relevant allergens. Adenosine was determined using an enzymatically coupled assay that produces ATP and is detected by luminescence. Uric acid was determined by ELISA. Exposure of cultured airway epithelial cells to Alternaria alternata extract caused significant nucleotide (NAD+ and ATP) release in the culture media. The addition of NAD+ to membranes prepared from WT mice resulted in faster generation of adenosine compared to membranes from CD38KO mice. Formation of adenosine from NAD+ affected UA and ATP concentrations, its main downstream molecules. Furthermore, NAD+ and adenosine concentrations in the bronchoalveolar lavage fluid decreased significantly following airway challenge with house-dust mite extract in WT but not in CD38KO mice. Thus, NAD+ is a significant source of adenosine and UA in the airways in mouse models of allergic airway disease, and the capacity for their generation from NAD+ is augmented by CD38, a major NADase with high affinity for NAD+. This novel non-canonical NAD+-adenosine-UA pathway that is triggered by allergens has not been previously described in the airways.
Assuntos
Adenosina/biossíntese , Hipersensibilidade/metabolismo , Pulmão/metabolismo , NAD/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Humanos , Hipersensibilidade/imunologia , Pulmão/imunologiaRESUMO
N6-methyladenosine (m6A) is the most common post-transcriptional modification of RNA in eukaryotes that regulates the post-transcriptional expression level of genes without changing the base sequence. The role of m6A in fungal keratitis has not yet been elucidated. Here, we aimed to identify m6A modification changes and their potential roles in fungal keratitis. The murine model of fungal keratitis was established by inoculating mice with Fusarium solani (F. solani). The overall m6A level was detected via an m6A RNA methylation assay kit. The expression levels of key m6A modification-related genes were estimated by quantitative real-time polymerase chain reaction (PCR). The expression and localization of METTL (methyltransferase like)3, the key component of the m6A methyltransferase complex, was determined by immunostaining and Western blotting (WB). Immunoprecipitation methylation microarray was used to describe the changes in m6A modification in F. solani-infected corneal tissue. The overall m6A level in corneal tissue on the 5th day in the F. solani-treated group was upregulated compared with that in the control group. The demethylase levels were unaltered, but the level of the methylase METTL3 was increased significantly after fungal infection. Additionally, differences were found in m6A modifications in 1137 mRNAs, of which 780 were hypermethylated and 357 were hypomethylated. To the best of our knowledge, the present work is the first investigation on the m6A modification profiles in experimental fungal keratitis, and it may provide a potential therapeutic target.
Assuntos
Adenosina/análogos & derivados , Córnea/metabolismo , Infecções Oculares Fúngicas/genética , Fusariose/genética , Fusarium/isolamento & purificação , Regulação da Expressão Gênica , Ceratite/genética , Adenosina/biossíntese , Adenosina/genética , Animais , Córnea/microbiologia , Córnea/patologia , Modelos Animais de Doenças , Infecções Oculares Fúngicas/metabolismo , Infecções Oculares Fúngicas/microbiologia , Fusariose/metabolismo , Fusariose/microbiologia , Ceratite/metabolismo , Ceratite/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Capuzes de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
Progressive diabetic nephropathy (DN) and loss of renal function correlate with kidney fibrosis. Crosstalk between TGF-ß and adenosinergic signaling contributes to the phenotypic transition of cells and to renal fibrosis in DN models. We evaluated the role of TGF-ß on NT5E gene expression coding for the ecto-5`-nucleotidase CD73, the limiting enzyme in extracellular adenosine production. We showed that high d-glucose may predispose HK-2 cells towards active transcription of the proximal promoter region of the NT5E gene while additional TGF-ß results in full activation. The epigenetic landscape of the NT5E gene promoter was modified by concurrent TGF-ß with occupancy by the p300 co-activator and the phosphorylated forms of the Smad2/3 complex and RNA Pol II. Transcriptional induction at NT5E in response to TGF-ß was earlier compared to the classic responsiveness genes PAI-1 and Fn1. CD73 levels and AMPase activity were concomitantly increased by TGF-ß in HK-2 cells. Interestingly, we found increased CD73 content in urinary extracellular vesicles only in diabetic patients with renal repercussions. Further, CD73-mediated AMPase activity was increased in the urinary sediment of DN patients. We conclude that the NT5E gene is a target of the profibrotic TGF-ß cascade and is a traceable marker of progressive DN.
Assuntos
5'-Nucleotidase/genética , Nefropatias Diabéticas/genética , Fibrose/genética , Fator de Crescimento Transformador beta/genética , Adenosina/biossíntese , Biomarcadores/metabolismo , Linhagem Celular , Nefropatias Diabéticas/patologia , Proteína p300 Associada a E1A/genética , Epigênese Genética/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose/patologia , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica , Humanos , Rim/metabolismo , Rim/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Nucleotidases/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genéticaRESUMO
Polymorphonuclear leukocytes (PMNs) are crucial for initial control of Streptococcus pneumoniae (pneumococcus) lung infection; however, as the infection progresses their persistence in the lungs becomes detrimental. Here we explored why the antimicrobial efficacy of PMNs declines over the course of infection. We found that the progressive inability of PMNs to control infection correlated with phenotypic differences characterized by a decrease in CD73 expression, an enzyme required for production of extracellular adenosine (EAD). EAD production by CD73 was crucial for the ability of both murine and human PMNs to kill S. pneumoniae. In exploring the mechanisms by which CD73 controlled PMN function, we found that CD73 mediated its antimicrobial activity by inhibiting IL-10 production. PMNs from wild-type mice did not increase IL-10 production in response to S. pneumoniae; however, CD73-/- PMNs up-regulated IL-10 production upon pneumococcal infection in vitro and during lung challenge. IL-10 inhibited the ability of WT PMNs to kill pneumococci. Conversely, blocking IL-10 boosted the bactericidal activity of CD73-/- PMNs as well as host resistance of CD73-/- mice to pneumococcal pneumonia. CD73/IL-10 did not affect apoptosis, bacterial uptake, and intracellular killing or production of antimicrobial neutrophil elastase and myeloperoxidase. Rather, inhibition of IL-10 production by CD73 was important for optimal reactive oxygen species (ROS) production by PMNs. ROS contributed to PMN antimicrobial function as their removal or detoxification impaired the ability of PMNs to efficiently kill S. pneumoniae. This study demonstrates that CD73 controls PMN antimicrobial phenotype during S. pneumoniae infection.
Assuntos
5'-Nucleotidase/fisiologia , Adenosina/fisiologia , Interleucina-10/biossíntese , Neutrófilos/enzimologia , Pneumonia Pneumocócica/imunologia , Streptococcus pneumoniae , 5'-Nucleotidase/biossíntese , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Adenosina/biossíntese , Transferência Adotiva , Adulto , Animais , Proteínas de Bactérias/genética , Grânulos Citoplasmáticos/enzimologia , Regulação para Baixo , Indução Enzimática , Líquido Extracelular , Feminino , Proteínas Ligadas por GPI/fisiologia , Humanos , Interleucina-10/genética , Elastase de Leucócito/biossíntese , Elastase de Leucócito/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Peroxidase/biossíntese , Peroxidase/genética , Pneumonia Pneumocócica/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Superóxido Dismutase/deficiência , Superóxido Dismutase/genética , Adulto JovemRESUMO
Cordyceps militaris is a mushroom species with high nutritive and medicinal values based on diverse bioactive metabolites. The contents of bioactive ingredients are indicative of the quality of commercially available fruit body of this fungus. Although the application of biotic elicitors has been an efficient strategy to induce the accumulation of valuable bioactive compounds in vivo, related research in C. militaris is rarely reported. In this study, five biotic elicitors in different concentrations (0.05, 0.5, 1, and 2 mg/mL), including chitosan (CHT), 2,4-dichlorophenoxyacetic acid (2,4-D), methyl jasmonate (MeJA), gibberellic acid (GA), and triacontanol (TRIA), were first introduced to enhance the production of 10 kinds of major bioactive components in the fruit body of C. militaris. Results showed that the effect of biotic elicitors on bioactive compounds in the fruit body of C. militaris was elicitor-specific and concentration-dependent. Overall, 1 mg/L CHT was considered the most favorable for the production of 10 bioactive ingredients in C. militaris fruit body, which could increase the content of protein, polysaccharides, polyphenol, triterpenoids, flavonoids, cordyceps acid, cordycepin, and anthocyanins by 20.38-, 1.41-, 0.7-, 0.47-, 11.90-, 1.09-, 0.34-, and 2.64-fold, respectively, compared with the control. The results of this study would provide an efficient strategy for the production of a superior quality fruit body of and contribute to further elucidation of the effects of biotic elicitors on metabolite accumulation in C. militaris.
Assuntos
Cordyceps/química , Cordyceps/efeitos dos fármacos , Extratos Vegetais/biossíntese , Reguladores de Crescimento de Plantas/farmacologia , Acetatos/farmacologia , Adenosina/análise , Adenosina/biossíntese , Agaricales/química , Agaricales/efeitos dos fármacos , Agaricales/metabolismo , Quitosana/farmacologia , Cordyceps/metabolismo , Ciclopentanos/farmacologia , Desoxiadenosinas/análise , Desoxiadenosinas/biossíntese , Carpóforos/química , Carpóforos/efeitos dos fármacos , Carpóforos/metabolismo , Giberelinas/farmacologia , Oxilipinas/farmacologia , Extratos Vegetais/química , Polissacarídeos/análise , Polissacarídeos/biossínteseRESUMO
The myo-inositol-1-phosphate synthase (MIPS) ortholog Ari2, which is encoded in the aristeromycin biosynthetic gene cluster, catalyzes the formation of five-membered cyclitol phosphate using d-fructose 6-phosphate (F6P) as a substrate. To understand the stereochemistry during the Ari2 reaction in vivo, we carried out feeding experiments with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]glucose in the aristeromycin-producing strain Streptomyces citricolor. We observed retention of the 2H atom of (6S)-d-[6-2H1]glucose and no incorporation of the 2H atom from (6R)-d-[6-2H1]glucose in aristeromycin. This indicates that Ari2 abstracts the pro-R proton at C6 of F6P after oxidation of C5-OH by nicotinamide adenine dinucleotide (NAD+) to generate the enolate intermediate, which then attacks the C2 ketone to form the C-C bond via aldol-type condensation. The reaction of Ari2 with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]F6P in vitro exhibited identical stereochemistry compared with that observed during the feeding experiments. Furthermore, analysis of the crystal structure of Ari2, including NAD+ as a ligand, revealed the active site of Ari2 to be similar to that of MIPS of Mycobacterium tuberculosis, supporting the similarity of the reaction mechanisms of Ari2 and MIPS.
Assuntos
Adenosina/análogos & derivados , Mio-Inositol-1-Fosfato Sintase/metabolismo , Adenosina/biossíntese , Adenosina/química , Modelos Moleculares , Mio-Inositol-1-Fosfato Sintase/química , Conformação Proteica , Estereoisomerismo , Streptomyces/enzimologiaRESUMO
The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC-AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC-AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.
Assuntos
Adenosina/análogos & derivados , Proteínas de Bactérias/química , RNA de Transferência/metabolismo , Adenosina/biossíntese , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Modelos Moleculares , Mutagênese , Conformação Proteica , RNA de Transferência/química , Thermotoga maritimaRESUMO
Adenosine, which is produced mainly by microbial fermentation, plays an important role in the therapy of cardiovascular disease and has been widely used as an antiarrhythmic agent. In this study, guanosine 5'-monophosphate (GMP) synthetase gene (guaA) was inactivated by gene-target manipulation to increase the metabolic flux from inosine 5'-monophosphate (IMP) to adenosine in B. subtilis A509. The resulted mutant M3-3 showed an increased adenosine production from 7.40 to 10.45 g/L, which was further enhanced to a maximum of 14.39 g/L by central composite design. As the synthesis of succinyladenosine monophosphate (sAMP) from IMP catalysed by adenylosuccinate synthetase (encoded by purA gene) is the rate-limiting step in adenosine synthesis, the up-regulated transcription level of purA was the potential underlying mechanism for the increased adenosine production. This work demonstrated a practical strategy for breeding B. subtilis strains for industrial nucleoside production.
Assuntos
Adenosina/genética , Adenilossuccinato Sintase/genética , Bacillus subtilis/genética , Carbono-Nitrogênio Ligases/genética , Adenosina/biossíntese , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/genética , Carbono-Nitrogênio Ligases/antagonistas & inibidores , Fermentação , Marcação de Genes , Inosina/genética , Inosina/metabolismo , Inosina Monofosfato/genética , Mutagênese Sítio-Dirigida , Mutação/genéticaRESUMO
Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2'-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.
Assuntos
Adenosina/análogos & derivados , Dioxigenase FTO Dependente de alfa-Cetoglutarato/fisiologia , RNA Nuclear Pequeno/biossíntese , Adenosina/biossíntese , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Processamento Alternativo , Animais , Células HEK293 , Humanos , Masculino , Metilação , Camundongos , Camundongos Knockout , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Nuclear Pequeno/metabolismoRESUMO
The radical S-adenosylmethionine (SAM) superfamily is currently the largest known enzyme family. These enzymes reductively cleave SAM to produce a highly reactive 5'-deoxyadenosyl (dAdo) radical, which abstracts a hydrogen from the substrate and initiates diverse reactions. The canonic dAdo radical-mediated hydrogen abstraction can be changed to radical addition reactions by using olefin-containing substrate analogues, which result in adenosylation reactions. Here we report investigation of the adenosylation reactions catalyzed by four radical SAM l-Tyr lyases (RSTLs), including HydG, FbiC, and two ThiH enzymes from different organisms. We show RSTLs have diverse substrate specificity, and ThiH from E. coli exhibits the highest substrate tolerance toward the tested substrates. We also show ThiH from Clostridium berjerinckii does not act on 4-amino-l-phenylalanine, but catalyzes adenosylation of the corresponding olefin-containing analogue, suggesting adenosylation may occur more easily than the canonic radical SAM reactions. Our study highlights the remarkable catalytic promiscuity of radical SAM enzyme and the potential in using these enzymes for the synthesis of nucleotide-containing compounds.