RESUMO
INTRODUCTION: Late-onset Alzheimer's disease (LOAD) is a complex neurodegenerative disease characterized by multiple progressive stages, glucose metabolic dysregulation, Alzheimer's disease (AD) pathology, and inexorable cognitive decline. Discovery of metabolic profiles unique to sex, apolipoprotein E (APOE) genotype, and stage of disease progression could provide critical insights for personalized LOAD medicine. METHODS: Sex- and APOE-specific metabolic networks were constructed based on changes in 127 metabolites of 656 serum samples from the Alzheimer's Disease Neuroimaging Initiative cohort. RESULTS: Application of an advanced analytical platform identified metabolic drivers and signatures clustered with sex and/or APOE É4, establishing patient-specific biomarkers predictive of disease state that significantly associated with cognitive function. Presence of the APOE É4 shifts metabolic signatures to a phosphatidylcholine-focused profile overriding sex-specific differences in serum metabolites of AD patients. DISCUSSION: These findings provide an initial but critical step in developing a diagnostic platform for personalized medicine by integrating metabolomic profiling and cognitive assessments to identify targeted precision therapeutics for AD patient subgroups through computational network modeling.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Masculino , Feminino , Humanos , Doença de Alzheimer/patologia , Medicina de Precisão , Doenças Neurodegenerativas/complicações , Genótipo , Apolipoproteínas E/genética , Apolipoproteína E4/genética , Redes e Vias MetabólicasRESUMO
Polyamines are essential for cell growth and proliferation. In plants and many bacteria, including Helicobacter pylori, the parent polyamine putrescine is only produced through the metabolism of N-carbamoylputrescine by N-carbamoylputrescine amidase (CPA). Thus, CPA is a crucial intermediate enzyme. Moreover, the absence of CPA in humans makes its presence in H. pylori a potential target for the development of new therapeutics against this pathogen. Despite this enzyme's presence in plants and bacteria, its function is not completely explored. Using structure-guided biochemical and biophysical studies on H. pylori CPA, we discovered an aromatic cluster containing four conserved tryptophans near the catalytic site and elucidated its role. Mutational studies revealed that they are individually vital to enzyme function. Unlike wild-type, which forms a hexamer, the Trp to Ala mutants only formed dimers. Interestingly, two other conserved residues, Gln155 and Asp278, interact with the tryptophan cluster and perform similar roles. Our results indicate that aromatic-aromatic and H-bonding contacts between the residues (Trp156-Trp273, Trp196-Gln155, and Trp153-Asp278) play a crucial role in stimulating activity through hexamer formation. Additionally, Trp156 is essential to generating a catalytically efficient hexamer. These results suggest dual roles for the tryptophans; in hexamer formation and in generating its functionally active form, thereby providing a mechanistic understanding into the role of the cluster. We also elucidated the catalytic roles of Glu43, Lys115, and Cys152, which are present at the active site. Our findings highlight, for the first time, the importance of a tryptophan cluster in H. pylori CPA that can be exploited to design therapeutic inhibitors.
Assuntos
Helicobacter pylori , Catálise , Domínio Catalítico , Humanos , Triptofano/metabolismoRESUMO
Coronaviruses are enveloped, non-segmented positive-sense RNA viruses with the largest genome among RNA viruses. Their genome contains a large replicase ORF which encodes nonstructural proteins (NSPs), structural, and accessory genes. NSP15 is a nidoviral RNA uridylate-specific endoribonuclease (NendoU) with C-terminal catalytic domain. The endoribonuclease activity of NSP15 interferes with the innate immune response of the host. Here, we screened Selleckchem Natural product database of the compounds against NSP15, and we found that thymopentin and oleuropein displayed highest binding energies. The binding of these molecules was further validated by molecular dynamic simulations that revealed them as very stable complexes. These drugs might serve as effective counter molecules in the reduction of virulence of this virus; may be more effective if treated in combination with replicase inhibitors. Future validation of both these inhibitors is worth the consideration for patients being treated for COVID-19. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42485-021-00059-w.
RESUMO
Entamoeba histolytica phosphoserine phosphatase (EhPSP), a regulatory enzyme in the serine biosynthetic pathway, is also a structural homolog of cofactor-dependent phosphoglycerate mutase (dPGM). However, despite sharing many of its catalytic residues with dPGM, EhPSP displays no significant mutase activity. In the current work, we determined a crystal structure of EhPSP in complex with 3-PGA to 2.5 Å resolution and observed striking differences between the orientation of 3-PGA bound to EhPSP and that to its other homologous structures. We also performed computational modeling and simulations of the intermediate 2,3-bisphosphoglyceric acid into the active site of EhPSP to better understand its mechanistic details. Based on these results and those of a similar study with the dPGMs from E. coli and B. pseudomallei, the affinity of EhPSP for 2,3-BPG was concluded to be lower than those of the other proteins. Moreover, a different set of 2,3-BPG interacting residues was observed in EhPSP compared to dPGMs, with all of the crucial interacting residues of dPGMs either missing or substituted with weakly interacting residues. This study has expanded our understanding, at the structural level, of the inability of EhPSP to catalyze the mutase reaction and has strengthened earlier conclusions indicating it to be a true phosphatase.
Assuntos
Entamoeba histolytica/enzimologia , Ácidos Glicéricos/química , Fosfoglicerato Mutase/química , Monoéster Fosfórico Hidrolases/química , Proteínas de Protozoários/química , Domínio Catalítico , Modelos Moleculares , Conformação Proteica , Alinhamento de SequênciaRESUMO
Mitosis is a cellular process that produces two identical progenies. Genome-wide transcription is believed to be silenced during mitosis. However, some transcription factors have been reported to associate with the mitotic chromatin to uphold a role in 'gene-bookmarking'. Here, we investigated the dynamic role of nuclear receptor SHP during cell cycle, and observed intermolecular interactions with PXR and ERα. This was reflected in altered subcellular localization, transcription function and mitotic chromatin behavior of these receptors. Subsequently, by in silico and live cell imaging approaches we identified the minimal domain(s) and crucial amino-acid residues required for such receptor-receptor interactions. It was apparent that both PXR/ERα interact with SHP to translocate cytoplasmic RFP-tagged SHP into the nucleus. In addition, during mitosis SHP interacted with some of the key nuclear receptors, altering partners, as well as, its own relationship with mitotic chromatin. SHP displaced a major fraction of PXR and ERα from the mitotic chromatin while promoted its own weak association reflected in its binding. Since SHP lacks DBD this association is attributed to receptor-receptor interactions rather than SHP-DNA interactions. The abrogation of PXR and ERα from the mitotic chromatin by SHP implies potential implications in regulation of gene bookmarking events in cellular development. Overall, it is concluded that intermolecular interactions between SHP and partner PXR/ERα result in attenuation of target promoter activities. It is proposed that SHP may act as an indirect physiological regulator and functions in a hog-tie manner by displacing the interacting transcription factor from gene regulatory sites.
Assuntos
Cromatina , Receptor alfa de Estrogênio , Mitose , Receptor de Pregnano X , Regiões Promotoras Genéticas , Transcrição Gênica , Transporte Ativo do Núcleo Celular/genética , Animais , Células COS , Chlorocebus aethiops , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Receptor de Pregnano X/química , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Leucyl-tRNA synthetases (LRS) catalyze the linkage of leucine with tRNALeu. A large insertion CP1 domain (Connective Polypeptide 1) in LRS is responsible for post-transfer editing of mis-charged aminoacyl-tRNAs. Here, we characterized the CP1 domain of Leishmania donovani, a protozoan parasite, and its role in editing activity and interaction with broad spectrum anti-fungal, AN2690. The deletion mutant of LRS, devoid of CP1 domain (LRS-CP1Δ) was constructed, followed by determination of its role in editing and aminoacylation. Binding of AN2690 and different amino acids with CP1 deletion mutant and full length LRS was evaluated using isothermal titration calorimetry (ITC) and molecular dynamics simulations. The recombinant LRS-CP1Δ protein did not catalyze the aminoacylation and the editing reaction when compared to full-length LRS. Thus, indicating that CP1 domain was imperative for both aminoacylation and editing activities of LRS. Binding studies with different amino acids indicated selectivity of isoleucine by CP1 domain over other amino acids. These studies also indicated high affinity of AN2690 with the editing domain. Molecular docking studies indicated that AN2690-CP1 domain complex was stabilized by hydrogen bonding and hydrophobic interactions resulting in high binding affinity between the two. Our data suggests CP1 is crucial for the function of L.donovani LRS.
Assuntos
Antiprotozoários/farmacologia , Compostos de Boro/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leishmania donovani/química , Leucina-tRNA Ligase/antagonistas & inibidores , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacologia , Antiprotozoários/química , Sítios de Ligação , Compostos de Boro/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Reposicionamento de Medicamentos , Expressão Gênica , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Leishmania donovani/enzimologia , Leishmania donovani/genética , Leucina-tRNA Ligase/química , Leucina-tRNA Ligase/genética , Leucina-tRNA Ligase/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Aminoacilação de RNA de Transferência/genéticaRESUMO
The l-cysteine is crucial for growth, survival, defense against oxidative stress, and pathogenesis of Entamoeba histolytica. The de novo biosynthesis of l-cysteine in E. histolytica, has a two-step pathway, where O-acetylserine sulfhydrylase (OASS) catalyses the last step by converting OAS to l-cysteine. This pathway is absent in humans and hence represents a promising target for novel therapeutics. E. histolytica expresses three isoforms of OASS and knockdown studies showed the importance of these enzymes for the survival of the pathogen. Here, we report the crystal structure of OASS isoform 3 from E. histolytica to 1.54 Å resolution. The active site geometries and kinetics of EhOASS3 and EhOASS1 structures were found to be very similar. Small-molecule libraries were screened against EhOASS3 and compounds were shortlisted based on the docking scores. F3226-1387 showed best inhibition with IC50 of 38 µM against EhOASS3 and was able to inhibit the growth of the organism to 72%.
Assuntos
Cisteína Sintase/antagonistas & inibidores , Entamoeba histolytica/citologia , Entamoeba histolytica/enzimologia , Inibidores Enzimáticos/farmacologia , Cristalografia por Raios X , Cisteína Sintase/química , Cisteína Sintase/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Entamoeba histolytica/crescimento & desenvolvimento , Inibidores Enzimáticos/química , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Entamoeba histolytica infection is highly prevalent in developing countries across the globe. The ATP synthesis in this pathogen is solely dependent on the glycolysis pathway where pyruvate kinase (Pyk) catalyzes the final reaction. Here, we have cloned, overexpressed and purified the pyruvate kinase (EhPyk) from E. histolytica. EhPyk is the shortest currently known Pyk till date as it contains only two of the three characterized domains when compared to the other homologues and our phylogenetic analysis places it on a distinct branch from the known type I/II Pyks. Our purification results suggested that it exists as a homodimer in solution. The kinetic characterization showed that EhPyk has maximum activity at pHâ¯7.5 where it exhibited Michaelis-Menten's kinetics for phosphoenolpyruvate with a Km of 0.23â¯mM, and it lost its activity at both the acidic pHâ¯4.0 and basic pHâ¯10.0. We also determined the key secondary structural elements of EhPyk at different pH values. MD simulation of EhPyk structure at different pH values suggested that it is most stable at pHâ¯7.0, while least stable at pHâ¯10.0 followed by pHâ¯4.0. Together, our computational simulations correlate well with the experimental studies. In summary, this study expands the current understanding of the EhPyk identified earlier in the amoebic genome and provides the first characterization of this bacterially expressed protein.
Assuntos
Entamoeba histolytica/enzimologia , Proteínas de Protozoários/química , Piruvato Quinase/química , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Fosfoenolpiruvato/química , Filogenia , Proteínas de Protozoários/genética , Piruvato Quinase/genéticaRESUMO
The Mycobacterium tuberculosis (Mtb) Rv2747 gene encodes for a functional protein known as ArgA, which plays an important role in the first step of the l-arginine biosynthesis pathway. ArgA transfers the acetyl group from the acetyl-CoA to either l-glutamate or l-glutamine, which are the known substrates. Here, we present two crystal structures of ArgA: one complexed with CoA and product bound N-acetylglutamine and the other complexed with acetyl-CoA and the inhibitor l-arginine at 2.3 and 3.0â¯Å resolution respectively. The Mtb ArgA protomer was found to have a "V" cleft and a "ß" bulge, archetypal of a classical GCN5-related N-acetyltransferase superfamily of proteins. The product bound form implies that ArgA can also acetylate l-glutamine like l-glutamate. The active site is strongly inhibited by l-arginine resulting in a closed conformation of ArgA and both l-arginine and N-acetylglutamine were found to occupy at the same active site. Together with structural analysis, molecular docking studies, microscale thermophoresis and enzyme inhibition assays, we conclude that l-glutamine, l-glutamate and l-arginine, all occupy at the same active site of ArgA. Furthermore in case of Mtb ArgA, l-arginine does not act as an allosteric inhibitor unlike other N-acetylglutamate synthase family of proteins.
Assuntos
Acetilcoenzima A/química , Acetiltransferases/química , Arginina/química , Proteínas de Bactérias/química , Ácido Glutâmico/química , Glutamina/química , Mycobacterium tuberculosis/química , Acetilcoenzima A/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/análogos & derivados , Glutamina/metabolismo , Cinética , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoAssuntos
Antineoplásicos/farmacologia , Moléculas de Adesão Celular/antagonistas & inibidores , Simulação por Computador , Terapia de Alvo Molecular , Antineoplásicos/química , Domínio Catalítico , Moléculas de Adesão Celular/química , Humanos , Imidazolidinas/química , Imidazolidinas/farmacologia , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , TermodinâmicaRESUMO
The helicase-primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58â Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase-primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB-DnaG complex. Also, MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Primase/metabolismo , DnaB Helicases/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cristalografia por Raios X , DNA Primase/química , DNA Primase/genética , DnaB Helicases/química , DnaB Helicases/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Mycobacterium tuberculosis/genética , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: Aminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the primary causative agent of visceral leishmaniasis. METHODOLOGY: Recombinant LdThrRS (rLdThrRS) was expressed in E. coli and purified. The kinetic parameters for rLdThrRS were determined. The subcellular localization of LdThrRS was done by immunofluorescence analysis. Heterozygous mutants of LdThrRS were generated in Leishmania promastigotes. These genetically manipulated parasites were checked for their proliferation, virulence, aminoacylation activity and sensitivity to the known ThrRS inhibitor, borrelidin. An in silico generated structural model of L. donovani ThrRS was compared to that of human. CONCLUSIONS: Recombinant LdThrRS displayed aminoacylation activity, and the protein is possibly localized to both the cytosol and mitochondria. The comparison of the 3D-model of LdThrRS to human ThrRS displayed considerable similarity. Heterozygous parasites showed restrictive growth phenotype and had attenuated infectivity. These heterozygous parasites were more susceptible to inhibition by borrelidin. Several attempts to obtain ThrRS homozygous null mutants were not successful, indicating its essentiality for the Leishmania parasite. Borrelidin showed a strong affinity for LdThrRS (KD: 0.04 µM) and was effective in inhibiting the aminoacylation activity of the rLdThrRS (IC50: 0.06 µM). Borrelidin inhibited the promastigotes (IC50: 21 µM) stage of parasites. Our data shows that LdThrRS is essential for L. donovani survival and is likely to bind with small drug-like molecules with strong affinity, thus making it a potential target for drug discovery efforts.
Assuntos
Leishmania donovani/enzimologia , Leishmaniose Visceral/parasitologia , Treonina-tRNA Ligase/genética , Sistemas de Liberação de Medicamentos , Escherichia coli/enzimologia , Escherichia coli/genética , Álcoois Graxos/farmacologia , Expressão Gênica , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Leishmania donovani/patogenicidade , Organismos Geneticamente Modificados , Filogenia , Domínios Proteicos , Transporte Proteico , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes , Deleção de Sequência , Treonina-tRNA Ligase/antagonistas & inibidores , Treonina-tRNA Ligase/isolamento & purificação , Treonina-tRNA Ligase/metabolismoRESUMO
Anxiety represents a public health problem consistently found to be the most prevalent class of mental disorders among people of all ages. Xanthones possess many biological properties, including neuroprotective, antioxidant or antidepressant-like. In this study, we aimed to investigate anxiolytic-like antidepressant and anticonvulsant properties of isolated xanthones from Swertia corymbosa. We evaluated anxiolytic-like activity of compounds 1-3 in the mouse elevated plus maze (EPM) and open field test (OF). We examined the influence on locomotor activity in mouse to determine if the effect observed in the actophotometer specific. We used step-through rotarod tests to evaluate the motor function and muscle grip. Compounds 1-3 significantly induced an increase in the number of entries into open arms and a decrease in time spent into closed arms at the dose of 50 mg/kg body weight (BW). The compounds also induced increase of rearing and decrease grooming at the doses of 25 and 50 mg/kg BW during the OF test. In addition, compounds induced a significant increase of time taken to enter at the center of the experimental set at the dose of 50 mg/kg BW during the open field test. The compounds 1-3 significantly delayed the onset as well as decreased the pentylenetetrazole and isoniazid-induced seizure tests. Compound 3 pretreatment significantly improved survivals in pentylenetetrazole and isoniazid-induced seizure tests. In silico studies reveal its possible mechanism of action shed on light to develop novel drugs against CNS disorders.
Assuntos
Simulação de Acoplamento Molecular , Neurofarmacologia , Swertia/química , Xantonas/química , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Ansiedade/tratamento farmacológico , Eletrochoque , Epilepsia/tratamento farmacológico , Isoniazida , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Atividade Motora/efeitos dos fármacos , Teste de Desempenho do Rota-Rod , Convulsões/tratamento farmacológico , Serotonina/química , Triazóis , Xantonas/isolamento & purificaçãoRESUMO
Urinary tract infections (UTIs) are diverse public health complication and caused by range of pathogens, however mostly Gram negative bacteria cause significant life threatening risks to different populations. The prevalence rate and antimicrobial resistance among the Gram negative uropathogens alarmed significantly heighten the economic burden of these infections. In this study, we investigated the antibiofilm efficiency of Pyrrolo [1,2-a] pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) extracted from endophytic actinomycetes Nocardiopsis sp. GRG 1 (KT235640) against P. mirabilis and E. coli. The extracted compound was characterized through TLC, HPLC, GC-MS, LC-MS and confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM). The compound, Pyrrolo [1,2-a] pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl) inhibits both bacterial biofilm formation as well as reduces the viability of preformed biofilms. Furthermore, CLSM image shows cell shrinkage, disorganized cell membrane and loss of viability. The SEM result also confirms the cell wall degradation in treated cells of the bacteria. Hence, the Pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) is active against P. mirabilis and E. coli.
Assuntos
Actinobacteria/química , Actinobacteria/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções Urinárias/microbiologia , Antibacterianos/isolamento & purificação , Biofilmes/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Chromobacterium/química , Chromobacterium/metabolismo , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Proteus mirabilis/citologia , Proteus mirabilis/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , beta-LactamasesRESUMO
The biosynthesis of UDP-N-acetylmuramic acid (UDP-MurNAc) by reduction of UDP-N-acetylglucosamine-enolpyruvate (UDP-GlcNAc-EP) in an NADPH and FAD-dependent reaction in bacteria is one of the key steps in peptidoglycan biosynthesis catalyzed by UDP-N-acetylglucosamine-enolpyruvate reductase (MurB). Here, we present the crystal structure of Mycobacterium tuberculosis MurB (MtbMurB) with FAD as the prosthetic group at 2.0Å resolution. There are six molecules in asymmetric unit in the form of dimers. Each protomer can be subdivided into three domains and the prosthetic group, FAD is bound in the active site between domain I and domain II. Comparison of MtbMurB structure with the structures of the Escherichia coli MurB (in complex with UDP-GlcNAc-EP) and Pseudomonas aeruginosa MurB (in complex with NADPH) showed all three structures share similar domain architecture and residues in the active site. The nicotinamide and the enol pyruvyl moieties are well aligned upon superimposition, both positioned in suitable position for hydride transfer to and from FAD. The comparison studies and MD simulations demonstrate that the two lobes of domain-III become more flexible. The substrates (NADPH and UDP-GlcNAc-EP) binding responsible for open conformation of MurB, suggesting that NADPH and UDP-GlcNAc-EP interactions are conformationally stable. Our findings provide a detail mechanism about the closed to open state by binding of NADPH and UDP-GlcNAc-EP induces the conformational changes of MurB structure that may trigger the MurB catalytic reaction.
Assuntos
Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/enzimologia , Uridina Difosfato N-Acetilglicosamina/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Mycobacterium tuberculosis/genética , NADP/química , NADP/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Homologia de Sequência de Aminoácidos , Uridina Difosfato N-Acetilglicosamina/química , Uridina Difosfato N-Acetilglicosamina/genética , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
Our study is to evaluate the potential bioactive compound of Nocardiopsis sp. GRG1 (KT235640) and its antibacterial activity against multi drug resistant strains (MDRS) on urinary tract infections (UTIs). Two brown algae samples were collected and were subjected to isolation of endophytic actinomycetes. 100 strains of actinomycetes were isolated from algal samples based on observation of morphology and physiological characters. 40 strains were active in antagonistic activity against various clinical pathogens. Among the strains, 10 showed better antimicrobial activity against MDRS on UTIs. The secondary metabolite of Nocardiopsis sp. GRG1 (KT235640) has showed tremendous antibacterial activity against UTI pathogens compared to other strains. Influence of various growth parameters were used for synthesis of secondary metabolites, such as optimum pH 7, incubation time 5-7 days, temperature (30 °C), salinity (5%), fructose and mannitol as the suitable carbon and nitrogen sources. At 100 µg/ml concentration MIC of Nocardiopsis sp. GRG1 (KT235640) showed highest percentage of inhibition against Proteus mirabilis (85%), and E.coli, Staphylococcus auerues, Psuedomonas aeroginasa, Enterobactor sp and Coagulinase negative staphylococci 78-85% respectively.
RESUMO
The Legionella pneumophila (Lp), human pathogen, causes severe and often fatal Legionnaires' disease, produces a major virulence factor, termed 'macrophage infectivity potentiator protein' (Mip), that is necessary for optimal multiplication of the bacteria within human alveolar macrophages. Mip exhibits peptidyl prolyl cis-trans isomerase (PPIase) activity, which can be inhibited by rapamycin and FK506. It was previously shown that substitutions at the catalytic residues, aspartate-142 position replaced to leucine-142 and tyrosine-185 position replaced to alanine-185 strongly reduces the PPIase activity of Mip proteins. Therefore, we aim to develop an in silico mutagenesis model for both important catalytic residues, validated the stability of the mutated model. Further, we have docked the known inhibitor rapamycin with Lp Mip (native) and mutants (D142L and Y185A) to analyze the conformational and binding mode. Electrostatic contributions and van der Waals interactions are the major driving forces for rapamycin binding and largely responsible for the binding differences between the Lp Mip (native and mutated) proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Simulação por Computador , Mutação/genética , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Sirolimo/farmacologia , Aminoácidos/genética , Sítios de Ligação , Ligação de Hidrogênio , Ligantes , Simulação de Acoplamento Molecular , Proteínas Mutantes/química , Ligação Proteica/efeitos dos fármacos , Conformação ProteicaRESUMO
The Legionella pneumophila (Lp), human pathogen causes severe and often fatal Legionnaires' disease, produces a major virulence factor, termed 'macrophage infectivity potentiator protein' (Mip), that is necessary for optimal multiplication of the bacteria within human alveolar macrophages. Mip exhibits peptidyl prolyl cistrans isomerase (PPIase) activity, which can be inhibited by Rapamycin and FK506. Mutation of Mip protein on catalytic residues at Aspartate-142 position replaced to Leucine-142 and Tyrosine-185 position replaced to Alanine-185 that strongly reduces the PPIase activity. Therefore, we aim to develop an in-silico mutagenesis model for both important catalytic residues, validated the stability of the mutated model. Further, we have docked to the known inhibitor rapamycin with Lp Mip (native) and mutants (D142L and Y185A) to analyze the conformational and binding model. For electrostatic contributions and VanderWaals interactions are the major driving force for rapamycin binding and largely responsible for the binding differences between the Lp Mip (native and mutated) proteins.
RESUMO
Pyruvate phosphate dikinase (PPDK) is the key enzyme essential for the glycolytic pathway in most common and perilous parasite Entamoeba histolytica. Inhibiting the function of this enzyme could control the wide spread of intestinal infections caused by Entamoeba histolytica in humans. With this objective, we modeled the three dimensional structure of the PPDK protein. We used templates with 51% identity and 67% similarity to employ homology-modeling approach. Stereo chemical quality of protein structure was validated by protein structure validation program PROCHECK and VERIFY3D. Experimental proof available in literature along with the in silico studies indicated Lys21, Arg91, Asp323, Glu325 and Gln337 to be the probable active sites in the target protein. Virtual screening was carried out using the genetic docking algorithm GOLD and a consensus scoring function X-Score to substantiate the prediction. The small molecule libraries (ChemDivision database, Diversity dataset, Kinase inhibitor database) were used for screening process. Along with the high scoring results, the interaction studies provided promising ligands for future experimental screening to inhibit the function of PPDK in Entamoeba histolytica. Further, the phylogeny study was carried out to assess the possibility of using the proposed ligands as inhibitors in related pathogens.
Assuntos
Entamoeba histolytica/enzimologia , Inibidores Enzimáticos/química , Modelos Moleculares , Preparações Farmacêuticas/química , Piruvato Ortofosfato Diquinase/química , Piruvatos/química , Algoritmos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Simulação por Computador , Bases de Dados Factuais , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Modelos Químicos , Conformação Molecular , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Conformação Proteica , Piruvato Ortofosfato Diquinase/antagonistas & inibidores , Piruvato Ortofosfato Diquinase/metabolismo , Homologia de Sequência de AminoácidosRESUMO
I-superfamily conotoxins have four-disulfide bonds with cysteine arrangement C-C-CC-CC-C-C, and they inhibit or modify ion channels of nerve cells. They have been characterized only recently and are relatively less well studied compared to other superfamily conotoxins. We have detected selective and sensitive sequence pattern for I-superfamily conotoxins. The availability of sequence pattern should be useful in protein family classification and functional annotation. We have built by homology modeling, a theoretical structural 3D model of ViTx from Conus virgo, a typical member of I-superfamily conotoxins. The modeling was based on the available 3D structure of Janus-atracotoxin-Hv1c of Janus-atracotoxin family whose members have been suggested as possible biopesticides. A study comparing the theoretically modeled structure of ViTx, with experimentally determined structures of other toxins, which share functional similarity with ViTx, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels.