RESUMO
The enzyme aspartate semialdehyde dehydrogenase (ASDH) plays a pivotal role in the amino acid biosynthesis pathway, making it an attractive target for the development of new antimicrobial drugs due to its absence in humans. This study aims to investigate the presence of ASDH in the filarial parasite Wolbachia endosymbiont of Brugia malayi (WBm) using both in vitro and in silico approaches. The size exclusion chromatography (SEC) and Native-PAGE analysis demonstrate that WBm-ASDH undergoes pH-dependent oligomerization and dimerization. To gain a deeper understanding of this phenomenon, the modelled monomer and dimer structures were subjected to pH-dependent dynamics simulations in various conditions. The results reveal that residues Val240, Gln161, Thr159, Tyr160, and Trp316 form strong hydrogen bond contacts in the intersurface area to maintain the structure in the dimeric form. Furthermore, the binding of NADP+ induces conformational changes, leading to an open or closed conformation in the structure. Importantly, the binding of NADP+ does not disturb either the dimerization or oligomerization of the protein, a finding confirmed through both in vitro and in silico analysis. These findings shed light on the structural characteristics of WBm-ASDH and offer valuable insights for the development of new inhibitors specific to WBm, thereby contributing to the development of potential therapies for filarial parasitic infections.
Assuntos
Aspartato-Semialdeído Desidrogenase , Brugia Malayi , Multimerização Proteica , Wolbachia , Brugia Malayi/enzimologia , Brugia Malayi/microbiologia , Concentração de Íons de Hidrogênio , Animais , Aspartato-Semialdeído Desidrogenase/metabolismo , Aspartato-Semialdeído Desidrogenase/química , Aspartato-Semialdeído Desidrogenase/genética , Wolbachia/enzimologia , Simulação de Dinâmica Molecular , Simulação por Computador , Simbiose , NADP/metabolismoRESUMO
Aspartate ß-semialdehyde dehydrogenase (ASADH) is a key enzyme in the biosynthesis of essential amino acids in microorganisms and some plants. Inhibition of ASADHs can be a potential drug target for developing novel antimicrobial and herbicidal compounds. This review covers up-to-date information about sequence diversity, ligand/inhibitor-bound 3D structures, potential inhibitors, and key pharmacophoric features of ASADH useful in designing novel and target-specific inhibitors of ASADH. Most reported ASADH inhibitors have two highly electronegative functional groups that interact with two key arginyl residues present in the active site of ASADHs. The structural information, active site binding modes, and key interactions between the enzyme and inhibitors serve as the basis for designing new and potent inhibitors against the ASADH family.
Assuntos
Aspartato-Semialdeído Desidrogenase , Inibidores Enzimáticos , Aspartato-Semialdeído Desidrogenase/química , Aspartato-Semialdeído Desidrogenase/metabolismo , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/químicaRESUMO
The emergence of drug-resistant tuberculosis (TB) constitutes a major challenge to TB control programmes. There is an urgent need to develop effective anti-TB drugs with novel mechanisms of action. Aspartate-semialdehyde dehydrogenase (ASADH) is the second enzyme in the aspartate metabolic pathway. The absence of the pathway in humans and the absolute requirement of aspartate in bacteria make ASADH a highly attractive drug target. In this study, we used ASADH coupled with Escherichia coli type III aspartate kinase (LysC) to establish a high-throughput screening method to find new anti-TB inhibitors. IMB-XMA0038 was identified as an inhibitor of MtASADH with an IC50 value of 0.59 µg/mL through screening. The interaction between IMB-XMA0038 and MtASADH was confirmed by surface plasmon resonance (SPR) assay and molecular docking analysis. Furthermore, IMB-XMA0038 was found to inhibit various drug-resistant MTB strains potently with minimal inhibitory concentrations (MICs) of 0.25-0.5 µg/mL. The conditional mutant strain MTB::asadh cultured with different concentrations of inducer (10-5 or 10-1 µg/mL pristinamycin) resulted in a maximal 16 times difference in MICs. At the same time, IMB-XMA0038 showed low cytotoxicity in vitro and vivo. In mouse model, it encouragingly declined the MTB colony forming units (CFU) in lung by 1.67 log10 dosed at 25 mg/kg for 15 days. In conclusion, our data demonstrate that IMB-XMA0038 is a promising lead compound against drug-resistant tuberculosis.
Assuntos
Antituberculosos/química , Aspartato-Semialdeído Desidrogenase/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Tuberculose/microbiologia , Animais , Antituberculosos/administração & dosagem , Aspartato-Semialdeído Desidrogenase/química , Aspartato-Semialdeído Desidrogenase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/administração & dosagem , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/genética , Tuberculose/tratamento farmacológicoRESUMO
This in silico work was carried out to reveal the proposed anti-fungal efficacy of some clove ingredient compounds against aspartate semialdehyde dehydrogenase, 6C8W and 6C85, enzymes from Blastomyces dermatitidis. The molecular docking simulation was implemented utilizing the Auto Dock 4.2. software. A set of 17 compounds were selected for this study, which is known to be active ingredients of Syzygium aromaticum crude and oil. The best docking scores associated with the Blastomyces dermatitidis enzymes 6C85 and 6C8W were for Maslinic acid and Oleanolic acid, followed by Stigmasterol and Campesterol. It was found that these compounds possess inhibitory potential against 6C85 and 6C8W and hence have anti-fungal efficacy. Maslinic acid and Oleanolic acid produced the strongest binding to 6C85 and 6C8W over the remaining bioactive compounds by forming H-bonds with some amino acids in these enzymes.
Assuntos
Antifúngicos/farmacologia , Aspartato-Semialdeído Desidrogenase/antagonistas & inibidores , Blastomyces/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Syzygium , Antifúngicos/isolamento & purificação , Aspartato-Semialdeído Desidrogenase/metabolismo , Blastomyces/enzimologia , Domínio Catalítico , Inibidores Enzimáticos/isolamento & purificação , Proteínas Fúngicas/metabolismo , Ligação de Hidrogênio , Ácido Oleanólico/isolamento & purificação , Ácido Oleanólico/farmacologia , Extratos Vegetais/isolamento & purificação , Conformação Proteica , Relação Estrutura-Atividade , Syzygium/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologiaRESUMO
The aspartate pathway, uniquely found in plants and microorganisms, offers novel potential targets for the development of new antimicrobial drugs. Aspartate semialdehyde dehydrogenase (ASADH) catalyzes production of a key intermediate at the first branch point in this pathway. Several fungal ASADH structures have been determined, but the prior crystallization conditions had precluded complex formation with enzyme inhibitors. The first inhibitor-bound and cofactor-bound structures of ASADH from the pathogenic fungi Blastomyces dermatitidis have now been determined, along with a structural and functional comparison to other ASADH family members. The structure of this new ASADH is similar to the other fungal orthologs, but with some critical differences in the orientation of some active site functional groups and in the subunit interface region. The presence of this bound inhibitor reveals the first details about inhibitor binding interactions, and the flexible orientation of its aromatic ring provides helpful insights into the design of potentially more potent and selective antifungal compounds.
Assuntos
Aspartato-Semialdeído Desidrogenase/química , Ácido Aspártico/química , Blastomyces/química , Coenzimas/química , Proteínas Fúngicas/química , NADP/química , Sequência de Aminoácidos , Aspartato-Semialdeído Desidrogenase/genética , Aspartato-Semialdeído Desidrogenase/metabolismo , Ácido Aspártico/metabolismo , Benzoquinonas/química , Benzoquinonas/metabolismo , Blastomyces/enzimologia , Domínio Catalítico , Clonagem Molecular , Coenzimas/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Simulação de Acoplamento Molecular , NADP/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , TermodinâmicaRESUMO
Aspartate ß-semialdehyde dehydrogenase (ASADH) is an enzyme involved in the diaminopimelate pathway of lysine biosynthesis. It is essential for the viability of many pathogenic bacteria and therefore has been the subject of considerable research for the generation of novel antibiotic compounds. This manuscript describes the first structure of ASADH from Francisella tularensis, the causative agent of tularemia and a potential bioterrorism agent. The structure was determined at 2.45â Å resolution and has a similar biological assembly to other bacterial homologs. ASADH is known to be dimeric in bacteria and have extensive interchain contacts, which are thought to create a half-sites reactivity enzyme. ASADH from higher organisms shows a tetrameric oligomerization, which also has implications for both reactivity and regulation. This work analyzes the apo form of F. tularensis ASADH, as well as the binding of the enzyme to its cofactor NADP+.
Assuntos
Aspartato-Semialdeído Desidrogenase/química , Proteínas de Bactérias/química , Francisella tularensis/enzimologia , Sequência de Aminoácidos , Aspartato-Semialdeído Desidrogenase/genética , Aspartato-Semialdeído Desidrogenase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Francisella tularensis/genética , Modelos Moleculares , NADP/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
Clostridium difficile is an enteric pathogen that causes approximately 20% to 30% of antibiotic-associated diarrhea. In recent years, there has been a substantial rise in the rate of C. difficile infections as well as the emergence of virulent and antibiotic resistant C. difficile strains. So, there is an urgent need for the identification of therapeutic potential targets and development of new drugs for the treatment and prevention of C. difficile infections. In the current study, we used a hybrid approach by combining sequence similarity-based approach and protein-protein interaction network topology-based approach to identify and characterize the potential drug targets of C. difficile. A total of 155 putative drug targets of C. difficile were identified and the metabolic pathway analysis of these putative drug targets using DAVID revealed that 46 of them are involved in 9 metabolic pathways. In-silico characterization of these proteins identified seven proteins involved in pathogen-specific peptidoglycan biosynthesis pathway. Three promising targets viz. homoserine dehydrogenase, aspartate-semialdehyde dehydrogenase and aspartokinase etc. were found to be involved in multiple enzymatic pathways of the pathogen. These 3 drug targets are of particular interest as they can be used for developing effective drugs against multi-drug resistant C. difficile strain 630 in the near future.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/metabolismo , Biologia Computacional/métodos , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Resistência a Múltiplos Medicamentos , Proteoma/metabolismo , Antibacterianos/farmacologia , Aspartato Quinase , Aspartato-Semialdeído Desidrogenase/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Fenômenos Bioquímicos , Clostridioides difficile/enzimologia , Clostridioides difficile/genética , Enterocolite Pseudomembranosa/tratamento farmacológico , Genes Essenciais/genética , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Peptidoglicano/biossíntese , Peptidoglicano/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteoma/genéticaRESUMO
Aspartate-semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of ß-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. The absence of this entire pathway in humans and other mammals will allow the selective targeting of pathogenic microorganisms for antimicrobial development. Here, the X-ray structure of a new form of ASADH from the pathogenic fungal species Aspergillus fumigatus has been determined. The overall structure of this enzyme is similar to those of its bacterial orthologs, but there are some critical differences both in biological assembly and in secondary-structural features that can potentially be exploited for the development of species-selective drugs with selective toxicity against infectious fungal organisms.
Assuntos
Aspartato-Semialdeído Desidrogenase/química , Ácido Aspártico/análogos & derivados , Aspergillus fumigatus/química , Proteínas Fúngicas/química , Sequência de Aminoácidos , Aspartato-Semialdeído Desidrogenase/genética , Aspartato-Semialdeído Desidrogenase/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Aspergillus fumigatus/enzimologia , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , NADP/química , NADP/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TermodinâmicaRESUMO
L-Aspartate-ß-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate pathway. In bacteria, ASADH is highly specific for the cofactor NADP(+) rather than NAD(+). Limited information on cofactor utilization is available, and neither the wild-type protein nor the available mutants could utilize NAD(+) efficiently. In this study, we identified several residues crucial for cofactor utilization by Escherichia coli ASADH (ecASADH) by mutating residues within the cofactor binding center. Among the investigated mutants, ecASADH-Q350N and ecASADH-Q350N/H171A, which exhibited markedly improved NAD(+) utilization, were further investigated by various biochemical approaches and molecular modeling. Relative to the wild type, the two mutants showed approximately 44-fold and 66-fold increases, respectively, in the constant kcat /Km of NAD(+). As desired, they could also utilize NADH efficiently to synthesize l-homoserine in cascade reactions in vitro.
Assuntos
Aspartato-Semialdeído Desidrogenase/genética , Aspartato-Semialdeído Desidrogenase/metabolismo , Escherichia coli/enzimologia , NAD/metabolismo , Aspartato-Semialdeído Desidrogenase/isolamento & purificação , Sítios de Ligação , Ativação Enzimática/genética , Escherichia coli/citologia , Escherichia coli/metabolismo , Modelos Moleculares , MutagêneseRESUMO
Aspartate-semialdehyde dehydrogenase (ASADH; EC 1.2.1.11) is a key enzyme in the biosynthesis of essential amino acids in prokaryotes and fungi, inhibition of ASADH leads to the development of novel antitubercular agents. In the present work, a combined structure and ligand-based pharmacophore modeling, molecular docking, and molecular dynamics (MD) approaches were employed to identify potent inhibitors of mycobacterium tuberculosis (Mtb)-ASADH. The structure-based pharmacophore hypothesis consists of three hydrogen bond acceptor (HBA), two negatively ionizable, and one positively ionizable center, while ligand-based pharmacophore consists of additional one HBA and one hydrogen bond donor features. The validated pharmacophore models were used to screen the chemical databases (ZINC and NCI). The screened hits were subjected to ADME and toxicity filters, and subsequently to the molecular docking analysis. Best-docked 25 compounds carry the characteristics of highly electronegative functional groups (-COOH and -NO2) on both sides and exhibited the H-bonding interactions with highly conserved residues Arg99, Arg249, and His256. For further validation of docking results, MD simulation studies were carried out on two representative compounds NSC51108 and ZINC04203124. Both the compounds remain bound to the key active residues of Mtb-ASADH during the MD simulations. These identified hits can be further used for lead optimization and in the design more potent inhibitors against Mtb-ASADH.
Assuntos
Aminoácidos Essenciais/química , Aspartato-Semialdeído Desidrogenase/química , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimologia , Aminoácidos Essenciais/biossíntese , Aspartato-Semialdeído Desidrogenase/genética , Aspartato-Semialdeído Desidrogenase/metabolismo , Ligação de Hidrogênio , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Células Procarióticas/enzimologia , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Aspartic acid semialdehyde dehydrogenase (ASADH) lies at the first branch point in the essential aspartic acid biosynthetic pathway that is found in bacteria and plants but is absent from animals. Mutations in the asadh gene encoding ASADH produce an inactive enzyme, which is lethal. Therefore, in this study, we investigated the hypothesis that ASADH represents a new anti-Mycobacterium tuberculosis (MTB) target. An asadh promoter-replacement mutant MTB, designated MTB::asadh, in which asadh gene expression is regulated by pristinamycin, was constructed to investigate the physiological functions of ASADH in the host bacteria. Bacterial growth was evaluated by monitoring OD600 and ASADH expression was analyzed by Western blotting. The results showed that the growth and survival of MTB::asadh was completely inhibited in the absence of the inducer pristinamycin. Furthermore, the growth of the mutant was rigorously dependent on the presence of the inducer in the medium. The starved mutant exhibited a marked reduction (approximately 80%) in the cell wall materials compared to the wild-type, in addition to obvious morphological differences that were apparent in scanning electron microscopy studies; however, with the addition of pristinamycin, the cell wall contents and morphology similar to those of the wild-type strain were recovered. The starved mutant also exhibited almost no pathogenicity in an in vitro model of infection using mouse macrophage J774A.1 cells. The mutant showed a concentration-dependent recovery of pathogenicity with the addition of the inducer. These findings implicate ASADH as a promising target for the development of novel anti-MTB drugs.
Assuntos
Antituberculosos/farmacologia , Aspartato-Semialdeído Desidrogenase/metabolismo , Mycobacterium tuberculosis/enzimologia , Animais , Western Blotting , Linhagem Celular , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Eletroforese em Gel de Ágar , Camundongos , Mutação/genética , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos TestesRESUMO
Aspartate-ß-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme.
Assuntos
Aspartato-Semialdeído Desidrogenase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Aspartato-Semialdeído Desidrogenase/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estrutura Molecular , Streptococcus pneumoniae/enzimologia , Relação Estrutura-AtividadeRESUMO
Aspartate ß-semialdehyde dehydrogenase (ASADH) is a key enzyme for the biosynthesis of essential amino acids and several important metabolites in microbes. Inhibition of ASADH enzyme is a promising drug target strategy against Mycobacterium tuberculosis (Mtb). In this work, in silico approach was used to identify potent inhibitors of Mtb-ASADH. Aspartyl ß-difluorophosphonate (ß-AFP), a known lead compound, was used to understand the molecular recognition interactions (using molecular docking and molecular dynamics analysis). This analysis helped in validating the computational protocol and established the participation of Arg99, Glu224, Cys130, Arg249, and His256 amino acids as the key amino acids in stabilizing ligand-enzyme interactions for effective binding, an essential feature is H-bonding interactions with the two arginyl residues at the two ends of the ligand. Best binding conformation of ß-AFP was selected as a template for shape-based virtual screening (ZINC and NCI databases) to identify compounds that competitively inhibit the Mtb-ASADH. The top rank hits were further subjected to ADME and toxicity filters. Final filter was based on molecular docking analysis. Each screened molecule carries the characteristics of the highly electronegative groups on both sides separated by an average distance of 6 Å. Finally, the best predicted 20 compounds exhibited minimum three H-bonding interactions with Arg99 and Arg249. These identified hits can be further used for designing the more potent inhibitors against ASADH family. MD simulations were also performed on two selected compounds (NSC4862 and ZINC02534243) for further validation. During the MD simulations, both compounds showed same H-bonding interactions and remained bound to key active residues of Mtb-ASADH.
Assuntos
Aspartato-Semialdeído Desidrogenase/química , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Aspartato-Semialdeído Desidrogenase/antagonistas & inibidores , Aspartato-Semialdeído Desidrogenase/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Simulação por Computador , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Ligação de Hidrogênio , Ligantes , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Organofosfonatos/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate ß-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors. Fragment-library screening has identified a new set of inhibitors that, while they do not resemble the substrates for this reaction, have been shown to bind at the active site of ASADH. Structure-guided development of these lead compounds has produced moderate inhibitors of the target enzyme, with some selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. However, many of these inhibitor analogs and derivatives have not yet achieved the expected enhanced affinity. Structural characterization of these enzyme-inhibitor complexes has provided detailed explanations for the barriers that interfere with optimal binding. Despite binding in the same active-site region, significant changes are observed in the orientation of these bound inhibitors that are caused by relatively modest structural alterations. Taken together, these studies present a cautionary tale for issues that can arise in the systematic approach to the modification of lead compounds that are being used to develop potent inhibitors.
Assuntos
Aspartato-Semialdeído Desidrogenase/antagonistas & inibidores , Aspartato-Semialdeído Desidrogenase/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Streptococcus pneumoniae/enzimologia , Vibrio cholerae/enzimologia , Aspartato-Semialdeído Desidrogenase/metabolismo , Ácido Aspártico/metabolismo , Vias Biossintéticas , Domínio Catalítico , Cólera/microbiologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Infecções Pneumocócicas/microbiologia , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Streptococcus pneumoniae/química , Streptococcus pneumoniae/metabolismo , Vibrio cholerae/química , Vibrio cholerae/metabolismoRESUMO
BACKGROUND: One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon. METHODS AND RESULTS: We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress. CONCLUSION: We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.
Assuntos
Adaptação Fisiológica/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Alanina Desidrogenase/genética , Alanina Desidrogenase/metabolismo , Aspartato-Semialdeído Desidrogenase/genética , Aspartato-Semialdeído Desidrogenase/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/farmacologia , Flagelina/genética , Flagelina/metabolismo , Deleção de Genes , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Cetol-Ácido Redutoisomerase/genética , Cetol-Ácido Redutoisomerase/metabolismo , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Óperon , Concentração Osmolar , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/deficiênciaRESUMO
Aggregation of aspartate-ß-semialdehyde dehydrogenase (ASD) was analyzed by applying modified Lumry-Eyring with nucleated polymerization (LENP) model. Intrinsic nucleation time scales were determined. In absence of glycerol, ASD undergoes concentration and time-dependent polymerization into low-molecular weight soluble aggregates and thereafter condensation into insoluble aggregates. In the presence of increasing solvent glycerol concentration, the aggregation becomes more and more nucleation dominated, with slower polymerization to low-molecular weights soluble aggregates, without any condensation into insoluble aggregates. Effective nucleus size as well as the number of monomers in each irreversible growth event were sensitive to the changes in solvent glycerol concentration. Glycerol-directed diminution of aggregation appears to be largely due to the inhibition of rearrangement (decreased nucleation rearrangement rate coefficient, K r,x ) because of compaction induced due to preferential hydration, thus, preventing the soluble aggregates from locking into irreversible soluble nuclei. Appreciably decreased K r,x (as compared to nucleation dissociation constant, K d,x ), appears to be responsible for increased nucleus size at higher solvent glycerol concentration. This study explains how modified LENP model can be applied to determine the predominant mechanism responsible for the diminution of aggregation by polyhydric alcohols (glycerol).
Assuntos
Aspartato-Semialdeído Desidrogenase/metabolismo , Glicerol/química , Aspartato-Semialdeído Desidrogenase/química , Aspartato-Semialdeído Desidrogenase/genética , Dicroísmo Circular , Polimerização , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solventes/química , Fatores de TempoRESUMO
Aspartate-semialdehyde dehydrogenase (Asd; ASADH; EC 1.2.1.11) is the enzyme that lies at the first branch point in the biosynthetic pathway of important amino acids including lysine and methionine and the cell-wall component diaminopimelate (DAP). The enzymatic reaction of ASADH is the reductive dephosphorylation of aspartyl-ß-phosphate (ABP) to aspartate ß-semialdehyde (ASA). Since the aspartate pathway is absolutely essential for the survival of many microbes and is absent in humans, the enzymes involved in this pathway can be considered to be potential antibacterial drug targets. In this work, the structure of ASADH from Mycobacterium tuberculosis H37Rv (Mtb-ASADH) has been determined in complex with glycerol and sulfate at 2.18 Å resolution and in complex with S-methyl-L-cysteine sulfoxide (SMCS) and sulfate at 1.95 Å resolution. The overall structure of Mtb-ASADH is similar to those of its orthologues. However, in the Mtb-ASADH-glycerol complex structure the glycerol molecule is noncovalently bound to the active-site residue Cys130, while in the Mtb-ASADH-SMCS complex structure the SMCS (Cys) is covalently linked to Cys130. The Mtb-ASADH-SMCS complex structurally mimics one of the intermediate steps in the proposed mechanism of ASADH enzyme catalysis. Comparison of the two complex structures revealed that the amino acids Glu224 and Arg249 undergo conformational changes upon binding of glycerol. Moreover, the structures reported here may help in the development of species-specific antibacterial drug molecules against human pathogens.
Assuntos
Aspartato-Semialdeído Desidrogenase/química , Mycobacterium tuberculosis/enzimologia , Aspartato-Semialdeído Desidrogenase/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Sulfatos/química , Sulfatos/metabolismoRESUMO
Microbes that have gained resistance against antibiotics pose a major emerging threat to human health. New targets must be identified that will guide the development of new classes of antibiotics. The selective inhibition of key microbial enzymes that are responsible for the biosynthesis of essential metabolites can be an effective way to counter this growing threat. Aspartate semialdehyde dehydrogenases (ASADHs) produce an early branch point metabolite in a microbial biosynthetic pathway for essential amino acids and for quorum sensing molecules. In this study, molecular modeling and docking studies were performed to achieve two key objectives that are important for the identification of new selective inhibitors of ASADH. First, virtual screening of a small library of compounds was used to identify new core structures that could serve as potential inhibitors of the ASADHs. Compounds have been identified from diverse chemical classes that are predicted to bind to ASADH with high affinity. Next, molecular docking studies were used to prioritize analogs within each class for synthesis and testing against representative bacterial forms of ASADH from Streptococcus pneumoniae and Vibrio cholerae. These studies have led to new micromolar inhibitors of ASADH, demonstrating the utility of this molecular modeling and docking approach for the identification of new classes of potential enzyme inhibitors.
Assuntos
Aspartato-Semialdeído Desidrogenase/antagonistas & inibidores , Inibidores Enzimáticos/química , Aspartato-Semialdeído Desidrogenase/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/síntese química , Cinética , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Streptococcus pneumoniae/enzimologia , Vibrio cholerae/enzimologiaRESUMO
The aspartate biosynthetic pathway provides essential metabolites for many important biological functions, including the production of four essential amino acids. As this critical pathway is only present in plants and microbes, any disruptions will be fatal to these organisms. An early pathway enzyme, l-aspartate-ß-semialdehyde dehydrogenase, produces a key intermediate at the first branch point of this pathway. Developing potent and selective inhibitors against several orthologs in the l-aspartate-ß-semialdehyde dehydrogenase family can serve as lead compounds for antibiotic development. Kinetic studies of two small molecule fragment libraries have identified inhibitors that show good selectivity against l-aspartate-ß-semialdehyde dehydrogenases from two different bacterial species, Streptococcus pneumoniae and Vibrio cholerae, despite the presence of an identical constellation of active site amino acids in this homologous enzyme family. Structural characterization of enzyme-inhibitor complexes have elucidated different modes of binding between these structurally related enzymes. This information provides the basis for a structure-guided approach to the development of more potent and more selective inhibitors.
Assuntos
Antibacterianos/química , Aspartato-Semialdeído Desidrogenase/antagonistas & inibidores , Inibidores Enzimáticos/química , Antibacterianos/síntese química , Antibacterianos/farmacologia , Aspartato-Semialdeído Desidrogenase/metabolismo , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Desenho de Fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Propionatos/química , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Especificidade por Substrato , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/enzimologiaRESUMO
In order to construct a conditional lethal Salmonella mutant, an arabinose-regulated recombinant genetic system was used. The Salmonella aspartate semialdehyde dehydrogenase (asd) gene was localized under the control of araC P(araBAD) in a plasmid to create the araC P(araBAD)::asd cassette. The cassette was cloned into a plasmid carrying a p15A replication origin to create the recombinant plasmid pMMP55. The growth of Salmonella MMP10 harboring pMMP55 was dependent on the presence of arabinose. In the presence of arabinose, the Asd deficiency due to chromosomal deletion of asd in the Salmonella host was complemented by the asd gene transcribed and translated under the P(araBAD) promoter and araBAD Shine-Dalgarno (SD) sequence in pMMP55. Growth inhibition of the strain was demonstrated by arabinose depletion in M9 minimal medium, indicating that the strain were unable to grow in an arabinose-limited environment. In addition, the analysis of a 50% lethal dose (LD50) using mice revealed that the strain MMP10 exhibited attenuation by approximately 100-fold relative to that of the unmodified strain. In conclusion, these data suggest that the araC P(araBAD)::asd system developed in this study can be used to construct conditional lethal Salmonella mutants for application as safe, live-attenuated Salmonella vaccines.