Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.538
Filtrar
1.
Nat Commun ; 11(1): 5874, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208736

RESUMO

Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5'-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription.


Assuntos
Betacoronavirus/química , Betacoronavirus/fisiologia , Replicação Viral , Betacoronavirus/genética , Betacoronavirus/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Viral/metabolismo , Relação Estrutura-Atividade , Transcrição Genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
2.
Sci Signal ; 13(651)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994211

RESUMO

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/química , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Virais/genética , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Fases de Leitura Aberta/genética , Pandemias , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
3.
Int J Biol Macromol ; 163: 1687-1696, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32980406

RESUMO

SARS-CoV-2 has caused COVID-19 outbreak with nearly 2 M infected people and over 100K death worldwide, until middle of April 2020. There is no confirmed drug for the treatment of COVID-19 yet. As the disease spread fast and threaten human life, repositioning of FDA approved drugs may provide fast options for treatment. In this aspect, structure-based drug design could be applied as a powerful approach in distinguishing the viral drug target regions from the host. Evaluation of variations in SARS-CoV-2 genome may ease finding specific drug targets in the viral genome. In this study, 3458 SARS-CoV-2 genome sequences isolated from all around the world were analyzed. Incidence of C17747T and A17858G mutations were observed to be much higher than others and they were on Nsp13, a vital enzyme of SARS-CoV-2. Effect of these mutations was evaluated on protein-drug interactions using in silico methods. The most potent drugs were found to interact with the key and neighbor residues of the active site responsible from ATP hydrolysis. As result, cangrelor, fludarabine, folic acid and polydatin were determined to be the most potent drugs which have potency to inhibit both the wild type and mutant SARS-CoV-2 helicase. Clinical data supporting these findings would be important towards overcoming COVID-19.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , Pneumonia Viral/tratamento farmacológico , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Betacoronavirus/enzimologia , Betacoronavirus/genética , Sítios de Ligação , Simulação por Computador , Infecções por Coronavirus/virologia , Aprovação de Drogas , Reposicionamento de Medicamentos , Ácido Fólico/farmacologia , Genoma Viral , Glucosídeos/farmacologia , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Mutação , Pandemias , Pneumonia Viral/virologia , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Estilbenos/farmacologia , Vidarabina/análogos & derivados , Vidarabina/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
4.
Life Sci ; 259: 118169, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32738360

RESUMO

AIMS: The recent outbreak of pandemic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led the world towards a global health emergency. Currently, no proper medicine or effective treatment strategies are available; therefore, repurposing of FDA approved drugs may play an important role in overcoming the situation. MATERIALS AND METHODS: The SARS-CoV-2 genome encodes for 2-O-methyltransferase (2'OMTase), which plays a key role in methylation of viral RNA for evading host immune system. In the present study, the protein sequence of 2'OMTase of SARS-CoV-2 was analyzed, and its structure was modeled by a comparative modeling approach and validated. The library of 3000 drugs was screened against the active site of 2'OMTase followed by re-docking analysis. The apo and ligand-bound 2'OMTase were further validated and analyzed by using molecular dynamics simulation. KEY FINDINGS: The modeled structure displayed the conserved characteristic fold of class I MTase family. The quality assessment analysis by SAVES server reveals that the modeled structure follows protein folding rules and of excellent quality. The docking analysis displayed that the active site of 2'OMTase accommodates an array of drugs, which includes alkaloids, antivirals, cardiac glycosides, anticancer, steroids, and other drugs. The redocking and MD simulation analysis of the best 5 FDA approved drugs reveals that these drugs form a stable conformation with the 2'OMTase. SIGNIFICANCE: The results suggested that these drugs may be used as potential inhibitors for 2'OMTase for combating the SARS-CoV-2 infection.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/antagonistas & inibidores , Pneumonia Viral/tratamento farmacológico , Antivirais/química , Antivirais/farmacologia , Biologia Computacional/métodos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Metilação/efeitos dos fármacos , Metiltransferases/química , Metiltransferases/metabolismo , Metiltransferases/ultraestrutura , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Pandemias , Pneumonia Viral/virologia , Homologia de Sequência de Aminoácidos
5.
Cell ; 182(6): 1560-1573.e13, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32783916

RESUMO

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.


Assuntos
Metiltransferases/química , RNA Helicases/química , Proteínas não Estruturais Virais/química , Replicação Viral , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Holoenzimas/química , Holoenzimas/metabolismo , Magnésio/metabolismo , Metiltransferases/metabolismo , Ligação Proteica , RNA Helicases/metabolismo , RNA Viral/química , Proteínas não Estruturais Virais/metabolismo
6.
Nat Commun ; 11(1): 3717, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709887

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.


Assuntos
Betacoronavirus/enzimologia , Metiltransferases/química , Proteínas não Estruturais Virais/química , Proteínas Virais Reguladoras e Acessórias/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Domínio Catalítico , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , Capuzes de RNA , Estabilidade de RNA , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
7.
Nat Commun ; 11(1): 3718, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709886

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.


Assuntos
Metiltransferases/química , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/química , Proteínas Virais Reguladoras e Acessórias/química , Betacoronavirus , Infecções por Coronavirus/virologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Difração de Raios X
8.
Nucleic Acids Res ; 48(18): 10034-10044, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32453412

RESUMO

S-adenosyl-l-methionine dependent methyltransferases catalyze methyl transfers onto a wide variety of target molecules, including DNA and RNA. We discuss a family of methyltransferases, those that act on the amino groups of adenine or cytosine in DNA, have conserved motifs in a particular order in their amino acid sequence, and are referred to as class beta MTases. Members of this class include M.EcoGII and M.EcoP15I from Escherichia coli, Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM), the MTA1-MTA9 complex from the ciliate Oxytricha, and the mammalian MettL3-MettL14 complex. These methyltransferases all generate N6-methyladenine in DNA, with some members having activity on single-stranded DNA as well as RNA. The beta class of methyltransferases has a unique multimeric feature, forming either homo- or hetero-dimers, allowing the enzyme to use division of labor between two subunits in terms of substrate recognition and methylation. We suggest that M.EcoGII may represent an ancestral form of these enzymes, as its activity is independent of the nucleic acid type (RNA or DNA), its strandedness (single or double), and its sequence (aside from the target adenine).


Assuntos
Evolução Molecular , Metiltransferases/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/enzimologia , Caulobacter crescentus/enzimologia , Escherichia coli/enzimologia , Humanos , Metiltransferases/classificação , Camundongos , Oxytricha/enzimologia , Conformação Proteica , Especificidade por Substrato
9.
Cells ; 9(5)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32443810

RESUMO

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/metabolismo , Replicação Viral/fisiologia , Animais , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Genoma Viral/genética , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Pandemias , Conformação Proteica em alfa-Hélice , RNA Helicases/química , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo
10.
J Plant Physiol ; 250: 153181, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32460036

RESUMO

Tetrandrine is the most effective small molecule that has been found to inhibit the Ebola virus. It is a typical bisbenzylisoquinoline alkaloid and is the main active ingredient in Stephania tetrandra. Metabolic engineering and synthetic biology are potential methods for efficient and rapid acquisition of tetrandrine. S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase (6OMT) is a rate-limiting step involved in the biosynthesis of tetrandrine. In this study, we identify S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase from S. tetrandra, which catalyzes the conversion of (S)-norcoclaurine to (S)-coclaurine. Four 6OMT-like genes were cloned from S. tetrandra. An in vitro enzyme assay showed that St6OMT1 could catalyze the conversion of (S)-norcoclaurine to produce (S)-coclaurine. St6OMT2 can catalyze the production of very few (S)-coclaurine molecules, accompanied by more by-products with m/z 300, compared to St6OMT1. The newly discovered 6OMTs will provide an optional genetic component for benzylisoquinoline alkaloid (BIA) synthetic biology research. This work will lay the foundation for the analysis of the biosynthetic pathway of tetrandrine in S. tetrandra.


Assuntos
Antivirais/metabolismo , Benzilisoquinolinas/metabolismo , Metiltransferases/genética , Proteínas de Plantas/genética , Stephania tetrandra/genética , Sequência de Aminoácidos , Metiltransferases/química , Metiltransferases/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Stephania tetrandra/enzimologia , Stephania tetrandra/metabolismo
11.
Nucleic Acids Res ; 48(9): 5094-5105, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32297938

RESUMO

Box C/D RNA protein complexes (RNPs) catalyze site-specific 2'-O-methylation of RNA with specificity determined by guide RNAs. In eukaryotic C/D RNP, the paralogous Nop58 and Nop56 proteins specifically associate with terminal C/D and internal C'/D' motifs of guide RNAs, respectively. We have reconstituted active C/D RNPs with recombinant proteins of the thermophilic yeast Chaetomium thermophilum. Nop58 and Nop56 could not distinguish between the two C/D motifs in the reconstituted enzyme, suggesting that the assembly specificity is imposed by trans-acting factors in vivo. The two C/D motifs are functionally independent and halfmer C/D RNAs can also guide site-specific methylation. Extensive pairing between C/D RNA and substrate is inhibitory to modification for both yeast and archaeal C/D RNPs. N6-methylated adenine at box D/D' interferes with the function of the coupled guide. Our data show that all C/D RNPs share the same functional organization and mechanism of action and provide insight into the assembly specificity of eukaryotic C/D RNPs.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas/metabolismo , Adenina/análogos & derivados , Chaetomium/genética , Humanos , Metilação , Conformação de Ácido Nucleico , RNA/metabolismo , Ribonucleoproteínas/química , Sulfolobus solfataricus
12.
Nucleic Acids Res ; 48(9): 5157-5168, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32266935

RESUMO

The N6-methyladenosine modification at position 43 (m6A43) of U6 snRNA is catalyzed by METTL16, and is important for the 5'-splice site recognition by U6 snRNA during pre-mRNA splicing. Human METTL16 consists of the N-terminal methyltransferase domain (MTD) and the C-terminal vertebrate conserved region (VCR). While the MTD has an intrinsic property to recognize a specific sequence in the distinct structural context of RNA, the VCR functions have remained uncharacterized. Here, we present structural and functional analyses of the human METTL16 VCR. The VCR increases the affinity of METTL16 toward U6 snRNA, and the conserved basic region in VCR is important for the METTL16-U6 snRNA interaction. The VCR structure is topologically homologous to the C-terminal RNA binding domain, KA1, in U6 snRNA-specific terminal uridylyl transferase 1 (TUT1). A chimera of the N-terminal MTD of METTL16 and the C-terminal KA1 of TUT1 methylated U6 snRNA more efficiently than the MTD, indicating the functional conservation of the VCR and KA1 for U6 snRNA biogenesis. The VCR interacts with the internal stem-loop (ISL) within U6 snRNA, and this interaction would induce the conformational rearrangement of the A43-containing region of U6 snRNA, thereby modifying the RNA structure to become suitable for productive catalysis by the MTD. Therefore, the MTD and VCR in METTL16 cooperatively facilitate the m6A43 U6 snRNA modification.


Assuntos
Metiltransferases/química , RNA Nuclear Pequeno/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Humanos , Metilação , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , Nucleotidiltransferases/química , Ligação Proteica , RNA Nuclear Pequeno/metabolismo
13.
J Mol Biol ; 432(9): 3018-3032, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32135193

RESUMO

The methyltransferases that belong to the SpoU-TrmD family contain trefoil knots in their backbone fold. Recent structural dynamic and binding analyses of both free and bound homologs indicate that the knot within the polypeptide backbone plays a significant role in the biological activity of the molecule. The knot loops form the S-adenosyl-methionine (SAM)-binding pocket as well as participate in SAM binding and catalysis. Knots contain both at once a stable core as well as moving parts that modulate long-range motions. Here, we sought to understand allosteric effects modulated by the knotted topology. Uncovering the residues that contribute to these changes and the functional aspects of these protein motions are essential to understanding the interplay between the knot, activation of the methyltransferase, and the implications in RNA interactions. The question we sought to address is as follows: How does the knot, which constricts the backbone as well as forms the SAM-binding pocket with its three distinctive loops, affect the binding mechanism? Using a minimally tied trefoil protein as the framework for understanding the structure-function roles, we offer an unprecedented view of the conformational mechanics of the knot and its relationship to the activation of the ligand molecule. Focusing on the biophysical characterization of the knot region by NMR spectroscopy, we identify the SAM-binding region and observe changes in the dynamics of the loops that form the knot. Importantly, we also observe long-range allosteric changes in flanking helices consistent with winding/unwinding in helical propensity as the knot tightens to secure the SAM cofactor.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Sítio Alostérico , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína
14.
Nat Commun ; 11(1): 1473, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193380

RESUMO

Caffeine is a major component of xanthine alkaloids and commonly consumed in many popular beverages. Due to its occasional side effects, reduction of caffeine in a natural way is of great importance and economic significance. Recent studies reveal that caffeine can be converted into non-stimulatory theacrine in the rare tea plant Camellia assamica var. kucha (Kucha), which involves oxidation at the C8 and methylation at the N9 positions of caffeine. However, the underlying molecular mechanism remains unclear. Here, we identify the theacrine synthase CkTcS from Kucha, which possesses novel N9-methyltransferase activity using 1,3,7-trimethyluric acid but not caffeine as a substrate, confirming that C8 oxidation takes place prior to N9-methylation. The crystal structure of the CkTcS complex reveals the key residues that are required for the N9-methylation, providing insights into how caffeine N-methyltransferases in tea plants have evolved to catalyze regioselective N-methylation through fine tuning of their active sites. These results may guide the future development of decaffeinated drinks.


Assuntos
Cafeína/metabolismo , Metiltransferases/metabolismo , Chá/enzimologia , Ácido Úrico/análogos & derivados , Sítios de Ligação , Vias Biossintéticas , Cafeína/química , Clonagem Molecular , Cristalografia por Raios X , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Metilação , Metiltransferases/química , Folhas de Planta/química , Proteínas Recombinantes/metabolismo , Chá/genética , Transcrição Genética , Ácido Úrico/química , Ácido Úrico/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(9): 4931-4941, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32075920

RESUMO

Paramyxoviruses are enveloped, nonsegmented, negative-strand RNA viruses that cause a wide spectrum of human and animal diseases. The viral genome, packaged by the nucleoprotein (N), serves as a template for the polymerase complex, composed of the large protein (L) and the homo-tetrameric phosphoprotein (P). The ∼250-kDa L possesses all enzymatic activities necessary for its function but requires P in vivo. Structural information is available for individual P domains from different paramyxoviruses, but how P interacts with L and how that affects the activity of L is largely unknown due to the lack of high-resolution structures of this complex in this viral family. In this study we determined the structure of the L-P complex from parainfluenza virus 5 (PIV5) at 4.3-Šresolution using cryoelectron microscopy, as well as the oligomerization domain (OD) of P at 1.4-Šresolution using X-ray crystallography. P-OD associates with the RNA-dependent RNA polymerase domain of L and protrudes away from it, while the X domain of one chain of P is bound near the L nucleotide entry site. The methyltransferase (MTase) domain and the C-terminal domain (CTD) of L adopt a unique conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase domain and near the likely exit site for the product RNA 5' end. Our study reveals a potential mechanism that mononegavirus polymerases may employ to switch between transcription and genome replication. This knowledge will assist in the design and development of antivirals against paramyxoviruses.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , Paramyxovirinae/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Cristalografia por Raios X , Genoma Viral , Metiltransferases/genética , Modelos Moleculares , Nucleoproteínas/química , Vírus da Parainfluenza 5/química , Paramyxovirinae/genética , Fosfoproteínas/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos
16.
Nat Commun ; 11(1): 864, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054833

RESUMO

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


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Heme/análogos & derivados , Metiltransferases/química , Metiltransferases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Eletroquímica , Ferroquelatase/química , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/biossíntese , Heme/química , Metiltransferases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidade por Substrato , Tetrapirróis/química , Tetrapirróis/metabolismo , Uroporfirinas/química , Uroporfirinas/metabolismo
17.
J Biol Chem ; 295(6): 1598-1612, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31914404

RESUMO

Benzylisoquinoline alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus (Nelumbo nucifera) is an ancient aquatic plant of medicinal value because of antiviral and immunomodulatory activities linked to its constituent BIAs. Although more than 30 BIAs belonging to the 1-benzylisoquinoline, aporphine, and bisbenzylisoquinoline structural subclasses and displaying a predominant R-enantiomeric conformation have been isolated from N. nucifera, its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two O-methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated NnOMT1-5 and encoding polypeptides sharing >40% amino acid sequence identity, were expressed in Escherichia coli Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-benzylisoquinoline 6-O-methyltransferase (6OMT) accepting both R- and S-substrates, whereas NnOMT5 is mainly a 7-O-methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for S-enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that O-methylation precedes BIA formation from 1-benzylisoquinoline intermediates. Km values for NnOMT1 and NnOMT5 were 20 and 13 µm for (R,S)-norcoclaurine and (S)-N-methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of alkaloid content, OMT activity in crude extracts, and OMT gene expression supported physiological roles for NnOMT1 and NnOMT5 in BIA metabolism, occurring primarily in young leaves and embryos of sacred lotus. In summary, our work identifies two OMTs involved in BIA metabolism in the medicinal plant N. nucifera.


Assuntos
Benzilisoquinolinas/metabolismo , Metiltransferases/metabolismo , Nelumbo/enzimologia , Proteínas de Plantas/metabolismo , Alcaloides/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Nelumbo/química , Nelumbo/genética , Nelumbo/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Alinhamento de Sequência
18.
Nucleic Acids Res ; 48(5): 2604-2620, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980825

RESUMO

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA-DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA-DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.


Assuntos
DNA Fúngico/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Moldes Genéticos , Elongação da Transcrição Genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biocatálise , DNA Fúngico/química , Cadeias de Markov , Metiltransferases/química , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , Nucleotídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , RNA Fúngico/biossíntese , Deleção de Sequência , Relação Estrutura-Atividade , Iniciação da Transcrição Genética
19.
Nucleic Acids Res ; 48(3): 1572-1582, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31919512

RESUMO

BCDIN3 domain containing RNA methyltransferase, BCDIN3D, monomethylates the 5'-monophosphate of cytoplasmic tRNAHis with a G-1:A73 mispair at the top of an eight-nucleotide-long acceptor helix, using S-adenosyl-l-methionine (SAM) as a methyl group donor. In humans, BCDIN3D overexpression is associated with the tumorigenic phenotype and poor prognosis in breast cancer. Here, we present the crystal structure of human BCDIN3D complexed with S-adenosyl-l-homocysteine. BCDIN3D adopts a classical Rossmann-fold methyltransferase structure. A comparison of the structure with that of the closely related methylphosphate capping enzyme, MePCE, which monomethylates the 5'-γ-phosphate of 7SK RNA, revealed the important residues for monomethyl transfer from SAM onto the 5'-monophosphate of tRNAHis and for tRNAHis recognition by BCDIN3D. A structural model of tRNAHis docking onto BCDIN3D suggested the molecular mechanism underlying the different activities between BCDIN3D and MePCE. A loop in BCDIN3D is shorter, as compared to the corresponding region that forms an α-helix to recognize the 5'-end of RNA in MePCE, and the G-1:A73 mispair in tRNAHis allows the N-terminal α-helix of BCDIN3D to wedge the G-1:A73 mispair of tRNAHis. As a result, the 5'-monophosphate of G-1 of tRNAHis is deep in the catalytic pocket for 5'-phosphate methylation. Thus, BCDIN3D is a tRNAHis-specific 5'-monomethylphosphate capping enzyme that discriminates tRNAHis from other tRNA species, and the structural information presented in this study also provides the molecular basis for the development of drugs against breast cancers.


Assuntos
Metiltransferases/ultraestrutura , RNA de Transferência de Histidina/ultraestrutura , RNA de Transferência/genética , S-Adenosil-Homocisteína/química , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Cristalografia por Raios X , Citoplasma/química , Citoplasma/genética , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Metilação , Metiltransferases/química , Metiltransferases/genética , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , RNA de Transferência/química , RNA de Transferência de Histidina/química , RNA de Transferência de Histidina/genética
20.
Nucleic Acids Res ; 48(1): e5, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31691820

RESUMO

RNA:5-methylcytosine (m5C) methyltransferases are currently the focus of intense research following a series of high-profile reports documenting their physiological links to several diseases. However, no methods exist which permit the specific analysis of RNA:m5C methyltransferases in cells. Herein, we described how a combination of biophysical studies led us to identify distinct duplex-remodelling effects of m5C on RNA and DNA duplexes. Specifically, m5C induces a C3'-endo to C2'-endo sugar-pucker switch in CpG RNA duplex but triggers a B-to-Z transformation in CpG DNA duplex. Inspired by these different 'structural signatures', we developed a m5C-sensitive probe which fluoresces spontaneously in response to m5C-induced sugar-pucker switch, hence useful for sensing RNA:m5C methyltransferase activity. Through the use of this probe, we achieved real-time imaging and flow cytometry analysis of NOP2/Sun RNA methyltransferase 2 (NSUN2) activity in HeLa cells. We further applied the probe to the cell-based screening of NSUN2 inhibitors. The developed strategy could also be adapted for the detection of DNA:m5C methyltransferases. This was demonstrated by the development of DNA m5C-probe which permits the screening of DNA methyltransferase 3A inhibitors. To our knowledge, this study represents not only the first examples of m5C-responsive probes, but also a new strategy for discriminating RNA and DNA m5C methyltransferase activity in cells.


Assuntos
DNA (Citosina-5-)-Metiltransferases/química , DNA/química , Corantes Fluorescentes/análise , Metiltransferases/química , Sondas Moleculares/análise , RNA/química , DNA/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo/métodos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Cinética , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Imagem Molecular/métodos , Sondas Moleculares/síntese química , Sondas Moleculares/metabolismo , Conformação de Ácido Nucleico , RNA/genética , RNA/metabolismo , Análise de Célula Única/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA