RESUMO
Isoprenoids are a diverse family of compounds that are synthesized from two isomeric compounds, isopentenyl diphosphate and dimethylallyl diphosphate. In most bacteria, isoprenoids are produced from the essential methylerythritol phosphate (MEP) pathway. The terminal enzymes of the MEP pathway IspG and IspH are [4Fe-4S] cluster proteins, and in Zymomonas mobilis, the substrates of IspG and IspH accumulate in cells in response to O2, suggesting possible lability of their [4Fe-4S] clusters. Here, we show using complementation assays in Escherichia coli that even under anaerobic conditions, Z. mobilis IspG and IspH are not as functional as their E. coli counterparts, requiring higher levels of expression to rescue viability. A deficit of the sulfur utilization factor (SUF) Fe-S cluster biogenesis pathway did not explain the reduced function of Z. mobilis IspG and IspH since no improvement in viability was observed in E. coli expressing the Z. mobilis SUF pathway or having increased expression of the E. coli SUF pathway. Complementation of single and double mutants with various combinations of Z. mobilis and E. coli IspG and IspH indicated that optimal growth required the pairing of IspG and IspH from the same species. Furthermore, Z. mobilis IspH conferred an O2-sensitive growth defect to E. coli that could be partially rescued by co-expression of Z. mobilis IspG. In vitro analysis showed O2 sensitivity of the [4Fe-4S] cluster of both Z. mobilis IspG and IspH. Altogether, our data indicate an important role of the cognate protein IspG in Z. mobilis IspH function under both aerobic and anaerobic conditions. IMPORTANCE: Isoprenoids are one of the largest classes of natural products, exhibiting diversity in structure and function. They also include compounds that are essential for cellular life across the biological world. In bacteria, isoprenoids are derived from two precursors, isopentenyl diphosphate and dimethylallyl diphosphate, synthesized primarily by the methylerythritol phosphate pathway. The aerotolerant Z. mobilis has the potential for methylerythritol phosphate pathway engineering by diverting some of the glucose that is typically efficiently converted into ethanol to produce isoprenoid precursors to make bioproducts and biofuels. Our data revealed the surprising finding that Z. mobilis IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway in part to evade the oxygen sensitivity of IspH.
Assuntos
Proteínas de Bactérias , Eritritol , Escherichia coli , Zymomonas , Zymomonas/metabolismo , Zymomonas/enzimologia , Zymomonas/genética , Eritritol/metabolismo , Eritritol/análogos & derivados , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Terpenos/metabolismo , OxirredutasesRESUMO
INTRODUCTION: Vibrio cholerae bacteria cause an infection characterized by acute diarrheal illness in the intestine. Cholera is sustained by people swallowing contaminated food or water. Even though symptoms can be mild, if untreated disease becomes severe and life-threatening, especially in low-income countries. AREAS COVERED: After a description of the most recent literature on the pathophysiology of this infection, we searched for patents and literature articles following the PRISMA guidelines, filtering the results disclosed from 2020 to present. Moreover, some innovative molecular targets (e.g., carbonic anhydrases) and pathways to counteract this rising problem were also discussed in terms of design, structure-activity relationships and structural analyses. EXPERT OPINION: This review aims to cover and analyze the most recent advances on the new druggable targets and bioactive compounds against this fastidious pathogen, overcoming the use of old antibiotics which currently suffer from high resistance rate.
Assuntos
Antibacterianos , Cólera , Desenho de Fármacos , Desenvolvimento de Medicamentos , Patentes como Assunto , Vibrio cholerae , Humanos , Antibacterianos/farmacologia , Cólera/tratamento farmacológico , Cólera/microbiologia , Vibrio cholerae/efeitos dos fármacos , Animais , Relação Estrutura-Atividade , Terapia de Alvo Molecular , Farmacorresistência Bacteriana , Diarreia/tratamento farmacológico , Diarreia/microbiologiaRESUMO
The incompressible smoothed particle hydrodynamics (ISPH) method is utilized for studying the circular rotations of three different structures, circular cylinder, rectangle and triangle centered in a circular cylinder cavity occupied by Al2O3 nanofluid. The novelty of this work is appearing in simulating the circular rotations of different solid structures on natural convection of a nanofluid-occupied a circular cylinder. The circular cylinder cavity is suspended by heterogeneous/homogeneous porous media. The embedded structures are taken as a circular cylinder, rectangle and triangle with equal areas. The first thermal condition considers the whole structure is heated, the second thermal condition considers the half of the structure is heated and the other is cooled and the third thermal condition considers the quarter of the structure is heated and the others are cooled. The outer boundary of cylinder cavity is cooled. Due to the small angular velocity ω=3.15 (low rotational speeds), then the natural convection case will be considered only. The results are representing the temperature, velocity fields. The simulations revealed that the presence of the inner hot/cold structures affects on the velocity distributions and temperature field inside a circular cylinder cavity. The triangle shape has introduced the highest temperature distributions and maximum values of the velocity fields compare to other shapes inside a circular cylinder cavity. The homogeneous porous level reduces the maximum values of velocity field by 25% compared to the heterogeneous porous level.
RESUMO
Soy allergens are susceptible to inducing allergic reactions in infants and young animals, which have an impact on the effective daily utilization of proteins. In this study, we used Alcalase-hydrolyzed instant soybean powder (ISP) to clarify the sensitization changes of instant soybean powder hydrolysates (ISPH), and we explored the assisted memory-enhancing effects. BALB/c mice in the ISPH group showed significant improvement in the allergy symptoms, with their allergy symptom scores decreasing to (1.57 ± 0.53) and their specific serum IgE and IgG1 binding capacity decreasing by 28.00 and 25.73% (P < 0.05), which suppressed the mast cell degranulation rate. Meanwhile, the plasma HIS and IL-4 levels decreased by 12.59 and 25.32%, and the plasma INF-γ and IL- 10 levels increased by 30.64 and 27.79%, which obviously regulated the imbalance of Th1/Th2 cells and attenuated the tissue damage (P < 0.05). Furthermore, ISPH improved behavioral characteristics, increased cholinergic system activity, reduced neuronal cell damage or apoptosis, and increased the number of Nissl bodies to help improve memory in Kunming mice (P < 0.05). In general, alcalase-hydrolyzed ISP had the dual effects of reducing allergenicity and aiding in memory improvement.
Assuntos
Hipersensibilidade Alimentar , Hipersensibilidade , Humanos , Camundongos , Lactente , Animais , Glycine max , Alérgenos , Pós , Imunoglobulina E , Subtilisinas , Camundongos Endogâmicos BALB C , Proteínas de SojaRESUMO
(E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) reductase (IspH) is a [4Fe-4S] cluster-containing enzyme, involved in isoprenoid biosynthesis as the final enzyme of the methylerythritol phosphate (MEP) pathway found in many bacteria and malaria parasites. In recent years, many studies have revealed that isoprenoid compounds are an alternative to petroleum-derived fuels. Thus, ecofriendly methods harnessing the methylerythritol phosphate pathway in microbes to synthesize isoprenoid compounds and IspH itself have received notable attention from researchers. In addition to its applications in the field of biosynthesis, IspH is considered to be an attractive drug target for infectious diseases such as malaria and tuberculosis due to its survivability in most pathogenic bacterium and its absence in humans. In this mini-review, we summarize previous reports that have systematically illuminated the fundamental and structural properties, substrate binding and catalysis, proposed catalytic mechanism, and novel catalytic activities of IspH. Potential bioengineering and biotechnological applications of IspH are also discussed.
RESUMO
The non-mevalonate or also called MEP pathway is an essential route for the biosynthesis of isoprenoid precursors in most bacteria and in microorganisms belonging to the Apicomplexa phylum, such as the parasite responsible for malaria. The absence of this pathway in mammalians makes it an interesting target for the discovery of novel anti-infectives. As last enzyme of this pathway, IspH is an oxygen sensitive [4Fe-4S] metalloenzyme that catalyzes 2H+/2e- reductions and a water elimination by involving non-conventional bioinorganic and bioorganometallic intermediates. After a detailed description of the discovery of the [4Fe-4S] cluster of IspH, this review focuses on the IspH mechanism discussing the results that have been obtained in the last decades using an approach combining chemistry, enzymology, crystallography, spectroscopies, and docking calculations. Considering the interesting druggability of this enzyme, a section about the inhibitors of IspH discovered up to now is reported as well. The presented results constitute a useful and rational help to inaugurate the design and development of new potential chemotherapeutics against pathogenic organisms.
Assuntos
Anti-Infecciosos/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxirredutases/metabolismo , Terpenos/química , Catálise , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Oxirredutases/fisiologiaRESUMO
Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form a redox system that is hypothesized to play a central role in the maintenance and function of the apicoplast organelle of malaria parasites. The Fd/FNR system provides reducing power to various iron-sulfur cluster (FeS)-dependent proteins in the apicoplast and is believed to help to maintain redox balance in the organelle. While the Fd/FNR system has been pursued as a target for antimalarial drug discovery, Fd, FNR, and the FeS proteins presumably reliant on their reducing power play an unknown role in parasite survival and apicoplast maintenance. To address these questions, we generated genetic deletions of these proteins in a parasite line containing an apicoplast bypass system. Through these deletions, we discovered that Fd, FNR, and certain FeS proteins are essential for parasite survival but found that none are required for apicoplast maintenance. Additionally, we addressed the question of how Fd and its downstream FeS proteins obtain FeS cofactors by deleting the FeS transfer proteins SufA and NfuApi. While individual deletions of these proteins revealed their dispensability, double deletion resulted in synthetic lethality, demonstrating a redundant role in providing FeS clusters to Fd and other essential FeS proteins. Our data support a model in which the reducing power from the Fd/FNR system to certain downstream FeS proteins is essential for the survival of blood-stage malaria parasites but not for organelle maintenance, while other FeS proteins are dispensable for this stage of parasite development. IMPORTANCE Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form one of the few known redox systems in the apicoplast of malaria parasites and provide reducing power to iron-sulfur (FeS) cluster proteins within the organelle. While the Fd/FNR system has been explored as a drug target, the essentiality and roles of this system and the identity of its downstream FeS proteins have not been determined. To answer these questions, we generated deletions of these proteins in an apicoplast metabolic bypass line (PfMev) and determined the minimal set of proteins required for parasite survival. Moving upstream of this pathway, we also generated individual and dual deletions of the two FeS transfer proteins that deliver FeS clusters to Fd and downstream FeS proteins. We found that both transfer proteins are dispensable, but double deletion displayed a synthetic lethal phenotype, demonstrating their functional redundancy. These findings provide important insights into apicoplast biochemistry and drug development.
Assuntos
Apicoplastos , Parasitos , Animais , Ferredoxinas/metabolismo , Parasitos/metabolismo , Plasmodium falciparum/metabolismo , Apicoplastos/metabolismo , NADP/metabolismo , Proteínas/metabolismo , Ferredoxina-NADP RedutaseRESUMO
BACKGROUND: Panax quinquefolius and Panax notoginseng are widely used and well known for their pharmacological effects. As main pharmacological components, saponins have different distribution patterns in the root tissues of Panax plants. METHODS: In this study, the representative ginsenosides were detected and quantified by desorption electrospray ionization mass spectrometry and high-performance liquid chromatography analysis to demonstrate saponin distribution in the root tissues of P. quinquefolius and P. notoginseng, and saponin metabolite profiles were analyzed by metabolomes to obtain the biomarkers of different root tissues. Finally, the transcriptome analysis was performed to demonstrate the molecular mechanisms of saponin distribution by gene profiles. RESULTS: There was saponin distribution in the root tissues differed between P. quinquefolius and P. notoginseng. Eight-eight and 24 potential biomarkers were detected by metabolome analysis, and a total of 340 and 122 transcripts involved in saponin synthesis that were positively correlated with the saponin contents (R > 0.6, P < 0.05) in the root tissues of P. quinquefolius and P. notoginseng, respectively. Among them, GDPS1, CYP51, CYP64, and UGT11 were significantly correlated with the contents of Rg1, Re, Rc, Rb2, and Rd in P. quinquefolius. UGT255 was markedly related to the content of R1; CYP74, CYP89, CYP100, CYP103, CYP109, and UGT190 were markedly correlated with the Rd content in P. notoginseng. CONCLUSIONS: These results provided the visual and quantitative profiles of and confirmed the pivotal transcripts of CYPs and UGTs regulating the saponin distribution in the root tissues of P. quinquefolius and P. notoginseng.
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IspH/LytB, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of the methylerythritol phosphate (MEP) pathway, a target for the development of new antimicrobial agents. This metalloenzyme converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, the synthesis of (S)-[4-2 H1 ]HMBPP and (R)-[4-2 H1 ]HMBPP is reported together with a detailed NMR analysis of the products formed after their respective incubation with E. coli IspH/LytB in the presence of the biological reduction system used by E. coli to reduce the [4Fe-4S] center. (S)-[4-2 H1 ]HMBPP was converted into [4-2 H1 ]DMAPP and (E)-[4-2 H1 ]IPP, whereas (R)-[4-2 H1 ]HMBPP yielded [4-2 H1 ]DMAPP and (Z)-[4-2 H1 ]IPP, hence providing the direct enzymatic evidence that the mechanism catalyzed by IspH/LytB involves a rotation of the CH2 OH group of the substrate to display it away from the [4Fe-4S].
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Oxirredutases/metabolismo , Fosfatos/metabolismo , Biocatálise , Organofosfatos/química , Organofosfatos/metabolismo , Oxirredução , Fosfatos/química , Especificidade por Substrato , Terpenos/química , Terpenos/metabolismoRESUMO
Panax notoginseng is famous for its important therapeutic effects. Saponins are bioactive compounds found in different parts and developmental stages of P. notoginseng plants. Thus, it is urgently to study saponins distribution in different parts and growth ages of P. notoginseng plants. In this study, potential biomarkers were found, and their chemical characteristic differences were revealed through metabolomic analysis. High-performance liquid chromatography data indicated the higher content of saponins (i.e., Rg1, Re, Rd, and Rb1) in the underground parts than that in the aerial parts. 20(S)-Protopanaxadiol saponins were mainly distributed in the aerial parts. Additionally, the total saponin content in the 3-year-old P. notoginseng plant (188.0 mg/g) was 1.4-fold higher than that in 2-year-old plant (130.5 mg/g). The transcriptomic analysis indicated the tissue-specific transcription expression of genes, namely, PnFPS, PnSS, PnSE1, PnSE2, and PnDS, which encoded critical synthases in saponin biosyntheses. These genes showed similar expression patterns among the parts of P. notoginseng plants. The expression levels of these genes in the flowers and leaves were 5.2fold higher than that in the roots and fibrils. These results suggested that saponins might be actively synthesized in the aerial parts and transformed to the underground parts. This study provides insights into the chemical and genetic characteristics of P. notoginseng to facilitate the synthesis of its secondary metabolites and a scientific basis for appropriate collection and rational use of this plant.
RESUMO
IspH, also called LytB, a protein involved in the biosynthesis of isoprenoids through the methylerythritol phosphate pathway, is an attractive target for the development of new antimicrobial drugs. Here, we report crystal structures of Escherichia coli IspH in complex with the two most potent inhibitors: (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate (TMBPP) and (E)-4-amino-3-methylbut-2-en-1-yl diphosphate (AMBPP) at 1.95 and 1.7â Å resolution, respectively. The structure of the E.â coli IspH:TMBPP complex exhibited two conformers of the inhibitor. This unexpected feature was exploited to design and evolve new antimicrobial candidates in silico.
Assuntos
Antibacterianos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Eritritol/análogos & derivados , Proteínas de Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Oxirredutases/química , Fosfatos Açúcares/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Eritritol/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismoRESUMO
The MEP pathway genes were modulated to investigate whether there were new rate-limiting steps and toxic intermediates in this pathway. Activating IspG led to significant decrease of cell growth and ß-carotene production. It was found that ispG overexpression led to accumulation of intermediate HMBPP, which seriously interfered with synthesis machinery of nucleotide and protein in Escherichia coli. Activation of the downstream enzyme IspH could solve HMBPP accumulation problem and eliminate the negative effects of ispG overexpression. In addition, intermediate MECPP accumulated in the starting strain, while balanced activation of IspG and IspH could push the carbon flux away from MECPP and led to 73% and 77% increase of ß-carotene and lycopene titer respectively. Our work for the first time identified HMBPP to be a cytotoxic intermediate in MEP pathway and demonstrated that balanced activation of IspG and IspH could eliminate accumulation of HMBPP and MECPP and improve isoprenoids production.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oxirredutases/metabolismo , Terpenos/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Oxirredutases/genéticaRESUMO
Isoprenoid biosynthesis is an important area for anti-infective drug development. One isoprenoid target is (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase (IspH), which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H+ /2e- reduction. IspH contains a 4 Fe-4 S cluster, and in this work, we first investigated how small molecules bound to the cluster by using HYSCORE and NRVS spectroscopies. The results of these, as well as other structural and spectroscopic investigations, led to the conclusion that, in most cases, ligands bound to IspH 4 Fe-4 S clusters by η1 coordination, forming tetrahedral geometries at the unique fourth Fe, ligand side chains preventing further ligand (e.g., H2 O, O2 ) binding. Based on these ideas, we used in silico methods to find drug-like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analogue with a Ki value of ≈500â nm against Pseudomonas aeruginosa IspH.
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Inibidores Enzimáticos/metabolismo , Hemiterpenos/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Organofosfatos/metabolismo , Compostos Organofosforados/metabolismo , Enxofre/metabolismo , Biologia Computacional , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação ProteicaRESUMO
(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) reductase (IspH, HDR or LytB) is an Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the last step of methylerythritol phosphate (MEP) pathway. The MEP pathway is known to produce 4-6:1 ratio of IPP and DMAPP mixture by the last enzyme, IspH. Plant IspH in plastids follows same catalytic mechanism as others, but GbIspH (Ginkgo biloba IspH) was reported to produce a mixture of IPP and DMAPP in a ratio of 16:1. Present catalytic mechanisms of IspH involve a common allyl anion intermediate, and the intramolecular proton transfer to the allyl moiety is considered as the key reaction step determining the product between IPP and DMAPP. The F212 residue in plant IspH was found as a potential amino acid residue that could mediate the proton transfer to the allyl anion intermediate before the product release. In this report, catalytic function of GbIspH F212 residue (H74 in E. coli), especially during the product formation in the active site, was studied by means of site-directed mutation. The product ratio of IPP/DMAPP was measured as 6.5 ± 0.1 for F212H GbIspH, and the value was close to the reported bacterial IspH having His residue on that specific position. Along with the other F212Y mutant, of which ratio was determined as 10.9 ± 0.1, the results strongly support that the Phe residue in plant IspH is the key amino acid residue that allows exclusive production of IPP in plant chloroplast.
Assuntos
Ginkgo biloba/química , Hemiterpenos/metabolismo , Compostos Organofosforados/metabolismo , Domínio Catalítico , Cloroplastos/metabolismo , Ginkgo biloba/metabolismo , Ferro/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Organofosfatos/metabolismo , Conformação Proteica , Estereoisomerismo , Sulfetos/metabolismoRESUMO
BACKGROUND: Bacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron-sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene. RESULTS: Bacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP(+)-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300% compared with the wild-type strain. CONCLUSIONS: IspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.
Assuntos
Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Hemiterpenos/biossíntese , Proteínas Ferro-Enxofre/metabolismo , Bacillus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Butadienos , Eletroforese em Gel de Poliacrilamida , Ferredoxinas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Hemiterpenos/química , Hemiterpenos/metabolismo , Proteínas Ferro-Enxofre/genética , Isomerismo , Mutagênese Sítio-Dirigida , Organofosfatos/química , Organofosfatos/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Pentanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Although several plasmids have been used in Listeria monocytogenes for generating mutants by allelic exchange, construction of L. monocytogenes mutants has been inefficient due to lack of effective selection markers for first and second recombination events. To address this problem, we have developed a new suicide plasmid, pHoss1, by using the pMAD plasmid backbone and anhydrotetracycline selection marker (secY antisense RNA) driven by an inducible Pxyl/tetO promoter. Expression of the secY antisense RNA eliminates merodiploids and selects for the loss of plasmid via a second allelic exchange, which enriches the number of mutants with deleted genes. To assess the effectiveness of pHoss1 for the generation of stable in-frame deletion mutations, we deleted the ispG and ispH genes of L. monocytogenes serotype 4b strain F2365. Results showed that identification of the second allelic exchange mutants was very efficient with 80-100% of the colonies yielding desired deletion mutants. L. monocytogenes' intestinal cell attachment was not altered when ispG and ispH genes were deleted. We expect that this new plasmid will be very useful for construction of marker-free deletion mutants in L. monocytogenes and in other Gram-positive bacteria, including Staphylococcus aureus and Bacillus cereus.
Assuntos
Genes Bacterianos , Listeria monocytogenes/genética , Mutação , Plasmídeos/genética , Aderência Bacteriana/genética , Ordem dos Genes , Teste de Complementação Genética , Fases de Leitura , Deleção de SequênciaRESUMO
The LytB/IspH protein catalyzes the last step of the methylerythritol phosphate (MEP) pathway which is used for the biosynthesis of essential terpenoids in most pathogenic bacteria. Therefore, the MEP pathway is a target for the development of new antimicrobial agents as it is essential for microorganisms, yet absent in humans. Substrate-free LytB has a special [4Fe-4S](2+) cluster with a yet unsolved structure. This motivated us to use synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) in combination with quantum chemical-molecular mechanical (QM/MM) calculations to gain more insight into the structure of substrate-free LytB. The apical iron atom of the [4Fe-4S](2+) is clearly linked to three water molecules. We additionally present NRVS data of LytB bound to its natural substrate, (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) and to the inhibitors (E)-4-amino-3-methylbut-2-en-1-yl diphosphate and (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Terpenos/metabolismo , Vias Biossintéticas , Cristalografia por Raios X , Difosfatos/química , Difosfatos/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear BiomolecularRESUMO
4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), also known as isoprenoid synthesis H (IspH) or lysis-tolerant B (LytB), catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and dimethylallyl diphosphate. The structure and reaction mechanism of IspH have been actively investigated in Escherichia coli but little is known in plants. Compared with the bacterial IspH, cyanobacterial and plant HDRs all contain an extra N-terminal conserved domain (NCD) that is essential for their function. Tyr72 in the NCD and several plant-specific residues around the central active site are critical for Arabidopsis HDR function. These results suggest that the structure and reaction mechanism of HDR/IspH may be different between plants and bacteria. The E. coli IspH is an iron-sulfur protein that is sensitive to oxygen. It is possible that the cyanobacterial HDR may independently evolve from the common ancestor of prokaryotes to obtain the NCD, which may protect the enzyme from high concentration of oxygen during photosynthesis.
Assuntos
Sequência Conservada , Oxigênio/metabolismo , Fotossíntese , Plantas/enzimologia , Sequência de Aminoácidos , Eritritol , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
In most of the pathogenic organisms including Plasmodium falciparum, isoprenoids are synthesized via MEP (MethylErythritol 4-Phosphate) pathway. LytB is the last enzyme of this pathway which catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Since the MEP pathway is not used by humans, it represents an attractive target for the development of new anti-malarial compounds or inhibitors. Here a systematic in silico study has been conducted to get an insight into the structure of Plasmodium lytB as well as its affinities towards different inhibitors. We used comparative modeling technique to predict the three-dimensional (3D) structure of Plasmodium LytB taking Escherichia coli LytB protein (PDB ID: 3KE8) as template and the model was subsequently refined through molecular dynamics (MD) simulation. A large ligand data-set containing diphospate group was subjected for virtual screening against the target using GOLD 5.2 program. Considering the mode of binding and affinities, 17 leads were selected on basis of binding energies in comparison to its substrate HMBPP (Gold.Chemscore.DG: -20.9734 kcal/mol). Among them, five were discarded because of their inhibitory activity towards other human enzymes. The rest 12 potential leads carry all the properties of any "drug like" molecule and the knowledge of Plasmodium LytB-inhibitory mechanism which can provide valuable support for the anti-malarial-inhibitor design in future.