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1.
J Enzyme Inhib Med Chem ; 34(1): 1414-1425, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31401901

RESUMO

The emergence of drug-resistant pathogenic bacteria is occurring due to the global overuse and misuse of ß-lactam antibiotics. Infections caused by some bacteria which secrete metallo-ß-lactamases (enzymes that inactivate ß-lactam antibiotics) are increasingly prevalent and have become a major worldwide threat to human health. These bacteria are resistant to ß-lactam antibiotics and MBL-inhibitor/ß-lactam antibiotic combination therapy can be a strategy to overcome this problem. So far, no clinically available inhibitors of metallo-ß-lactamases (MBLs) have been reported. In this study, L-benzyl tyrosine thiol carboxylic acid analogues (2a-2k) were synthesized after the study of computational simulation by adding of methyl, chloro, bromo and nitro groups to the benzyl ring for investigation of SAR analysis. Although the synthesized molecules 2a-k shows the potent inhibitory effects against metallo-ß-lactamase (IMP-1) with the range of Kic values of 1.04-4.77 µM, they are not as potent as the candidate inhibitor.


Assuntos
Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologia , Compostos de Sulfidrila/química , Tirosina/química , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Ácidos Carboxílicos/química , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Espectroscopia de Prótons por Ressonância Magnética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/enzimologia , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/química
2.
J Agric Food Chem ; 67(31): 8581-8589, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31321975

RESUMO

Intermediates in aromatic amino acid biosynthesis can serve as substrates for the synthesis of bioactive compounds. In this study we used two intermediates in the shikimate pathway of Escherichia coli, chorismate and anthranilate, to synthesize three bioactive compounds: 4-hydroxycoumarin (4-HC), 2,4-dihydroxyquinoline (DHQ), and 4-hydroxy-1-methyl-2(1H)-quinolone (NMQ). We introduced genes for the synthesis of salicylic acid from chorismate to supply the substrate for 4-HC and the gene encoding N-methyltransferase for the synthesis of N-methylanthranilate from anthranilate. Polyketide synthases and coenzyme (Co)A ligases were tested to determine the optimal combination of genes for the synthesis of each compound. We also tested several constructs and identified the best one for increasing levels of endogenous substrates for chorismate, anthranilate, and malonyl-CoA. With the use of these strategies, 255.4 mg/L 4-HC, 753.7 mg/L DHQ, and 17.5 mg/L NMQ were synthesized. This work provides a basis for the synthesis of diverse coumarin and quinoline derivatives with potential medical applications.


Assuntos
4-Hidroxicumarinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Policetídeo Sintases/genética , Quinolinas/metabolismo , 4-Hidroxicumarinas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Corísmico/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Photorhabdus/enzimologia , Photorhabdus/genética , Policetídeo Sintases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Quinolinas/química , ortoaminobenzoatos/metabolismo
3.
J Microbiol ; 57(8): 704-710, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31187416

RESUMO

KatA is the major catalase required for hydrogen peroxide (H2O2) resistance and acute virulence in Pseudomonas aeruginosa PA14, whose transcription is governed by its dual promoters (katAp1 and katAp2). Here, we observed that KatA was not required for acute virulence in another wild type P. aeruginosa strain, PAO1, but that PAO1 exhibited higher KatA expression than PA14 did. This was in a good agreement with the observation that PAO1 was more resistant than PA14 to H2O2 as well as to the antibiotic peptide, polymyxin B (PMB), supposed to involve reactive oxygen species (ROS) for its antibacterial activity. The higher KatA expression in PAO1 than in PA14 was attributed to both katAp1 and katAp2 transcripts, as assessed by S1 nuclease mapping. In addition, it was confirmed that the PMB resistance is attributed to both katAp1 and katAp2 in a complementary manner in PA14 and PAO1, by exploiting the promoter mutants for each -10 box (p1m, p2m, and p1p2m). These results provide an evidence that the two widely used P. aeruginosa strains display different virulence mechanisms associated with OxyR and Anr, which need to be further characterized for better understanding of the critical virulence pathways that may differ in various P. aeruginosa strains.


Assuntos
Proteínas de Bactérias/genética , Catalase/genética , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Polimixina B/metabolismo , Pseudomonas aeruginosa/enzimologia , Antibacterianos/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/patogenicidade , Virulência
4.
J Microbiol Biotechnol ; 29(6): 839-844, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154751

RESUMO

Anthranilate derivatives have been used as flavoring and fragrant agents for a long time. Recently, these compounds are gaining attention due to new biological functions including antinociceptive and analgesic activities. Three anthranilate derivatives, N-methylanthranilate, methyl anthranilate, and methyl N-methylanthranilate were synthesized using metabolically engineered stains of Escherichia coli. NMT encoding N-methyltransferase from Ruta graveolens, AMAT encoding anthraniloyl-coenzyme A (CoA):methanol acyltransferase from Vitis labrusca, and pqsA encoding anthranilate coenzyme A ligase from Pseudomonas aeruginosa were cloned and E. coli strains harboring these genes were used to synthesize the three desired compounds. E. coli mutants (metJ, trpD, tyrR mutants), which provide more anthranilate and/or S-adenosyl methionine, were used to increase the production of the synthesized compounds. MS/MS analysis was used to determine the structure of the products. Approximately, 185.3 µM N-methylanthranilate and 95.2 µM methyl N-methylanthranilate were synthesized. This is the first report about the synthesis of anthranilate derivatives in E. coli.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , ortoaminobenzoatos/metabolismo , Vias Biossintéticas , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/metabolismo , Ruta/enzimologia , Ruta/genética , Vitis/enzimologia , Vitis/genética , ortoaminobenzoatos/química
5.
BMC Infect Dis ; 19(1): 565, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253101

RESUMO

BACKGROUND: To detect carbapenemase-producing Gram-negative bacteria in bacterial laboratories at medical settings, a new immunochromatographic assay for New Delhi metallo-ß-lactamases (NDMs) was developed. METHODS: The immunochromatographic assay for New Delhi metallo-ß-lactamases producers was developed using rat monoclonal antibodies against NDMs. The assessment was performed using 350 isolates of Gram-negative bacteria, including Acinetobacter baumannii (51 isolates), Enterobacteriaceae (163 isolates), and Pseudomonas aeruginosa (136 isolates) obtained from 2015 to 2017 in medical settings in Myanmar. Of them, 302 isolates were resistant to carbapenems, including imipenem and/or meropenem. The blaNDM genes were identified by PCR and sequencing. RESULTS: Of the 350 clinical isolates tested, 164 (46.9%) (60 isolates of Escherichia coli, 51 isolates of Klebsiella pneumoniae, 25 isolates of Enterobacter cloacae, 23 isolates of P. aeruginosa, and 5 isolates of A. baumannii) were positive on this assay, and all the positive isolates harbored genes encoding NDM-1, - 4, - 5 and - 7. The remaining 186 (53.1%) isolates negative on the assay did not harbor genes encoding NDMs. The assay had a specificity of 100% and a sensitivity of 100%. The assessment revealed that more than 90% of carbapenem-resistant Enterobacteriaceae produced NDMs. CONCLUSIONS: The immunochromatographic assay is an easy-to-use and reliable kit for detection of NDMs-producing Gram-negative bacteria. The assay revealed that NDM-producing Enterobacteriaceae isolates are wide-spread in medical settings in Myanmar.


Assuntos
Bactérias Gram-Negativas/isolamento & purificação , Imunoensaio/métodos , beta-Lactamases/imunologia , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/isolamento & purificação , Animais , Antibacterianos/farmacologia , Anticorpos Monoclonais/imunologia , Farmacorresistência Bacteriana , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Humanos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Mianmar , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , Ratos , beta-Lactamases/genética , beta-Lactamases/metabolismo
6.
Nat Commun ; 10(1): 2853, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253808

RESUMO

Plant innate immunity restricts growth of bacterial pathogens that threaten global food security. However, the mechanisms by which plant immunity suppresses bacterial growth remain enigmatic. Here we show that Arabidopsis thaliana secreted aspartic protease 1 and 2 (SAP1 and SAP2) cleave the evolutionarily conserved bacterial protein MucD to redundantly inhibit the growth of the bacterial pathogen Pseudomonas syringae. Antibacterial activity of SAP1 requires its protease activity in planta and in vitro. Plants overexpressing SAP1 exhibit enhanced MucD cleavage and resistance but incur no penalties in growth and reproduction, while sap1 sap2 double mutant plants exhibit compromised MucD cleavage and resistance against P. syringae. P. syringae lacking mucD shows compromised growth in planta and in vitro. Notably, growth of ΔmucD complemented with the non-cleavable MucDF106Y is not affected by SAP activity in planta and in vitro. Our findings identify the genetic factors and biochemical process underlying an antibacterial mechanism in plants.


Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Serina Endopeptidases/metabolismo , Arabidopsis/imunologia , Proteínas de Bactérias/genética , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidases/genética
7.
Int J Infect Dis ; 84: 143-150, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31204002

RESUMO

OBJECTIVES: Carbapenem resistance in Pseudomonas aeruginosa is growing and results from variable mechanisms. The objectives of the current study were to investigate mechanisms of carbapenem resistance and genetic relatedness of P. aeruginosa isolates recovered in Dubai hospitals. METHODS: From June 2015 through June 2016, carbapenem-nonsusceptible P. aeruginosa were collected from 4 hospitals in Dubai, and subjected to antimicrobial susceptibility testing, molecular investigation of carbapenemases by PCR-sequencing, analysis of outer membrane porin OprD2 and multidrug efflux channel MexAB-OprM levels by qPCR, and fingerprinting by ERIC-PCR. RESULTS: Out of 1969 P. aeruginosa isolated during the study period, 471 (23.9%) showed reduced carbapenem susceptibility. Of these, 37 were analyzed and 32% of them produced VIM-type metallo-ß-lactamases, including VIM-2, VIM-30, VIM-31, and VIM-42, while GES-5 and GES-9 co-existed with VIM in 5.4% of isolates. Outer membrane impermeability was observed in 73% of isolates and 75.6% displayed overproduced MexAB-OprM. ERIC-PCR revealed one large clone including most carbapenemase-producing isolates indicating clonal dissemination. CONCLUSION: This is the first study on carbapenem-nonsusceptible P. aeruginosa from Dubai, incriminating VIM production as well as outer membrane permeability and efflux systems as resistance mechanisms. Further studies on carbapenem-nonsusceptible P. aeruginosa in Dubai are warranted for containment of such health hazard.


Assuntos
Proteínas de Bactérias/fisiologia , Carbapenêmicos/farmacologia , Porinas/fisiologia , Pseudomonas aeruginosa/efeitos dos fármacos , beta-Lactamases/fisiologia , Permeabilidade da Membrana Celular , Estudos Transversais , Farmacorresistência Bacteriana , Humanos , Pseudomonas aeruginosa/enzimologia
8.
Curr Pharm Biotechnol ; 20(6): 497-505, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31038060

RESUMO

BACKGROUND: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. OBJECTIVES: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. METHODS: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. RESULTS: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. CONCLUSION: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.


Assuntos
Biotecnologia/métodos , Enzimas Imobilizadas/metabolismo , Ouro/química , Lipase/metabolismo , Nanopartículas Metálicas/química , Biocatálise , Enzimas Imobilizadas/genética , Escherichia coli/genética , Lipase/genética , Plasmídeos , Pseudomonas aeruginosa/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
J Med Microbiol ; 68(6): 952-956, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31107204

RESUMO

Exploiting the immunosuppressive, analgesic and highly addictive properties of morphine could increase the success of a bacterial pathogen. Therefore, we performed sequence similarity searches for two morphine biosynthesis demethylases in bacteria. For thebaine 6-O-demethylase and codeine O-demethylase, we found strong alignments to three (Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) of the six ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) that are commonly associated with drug resistance and nosocomial infections. Expression of the aligned sequence found in P. aeruginosa (NP_252880.1/PA4191) is upregulated in isolates obtained from cystic fibrosis patients. Our findings provide putative mechanistic targets for understanding the role of morphine in pathogenicity.


Assuntos
Acinetobacter baumannii/enzimologia , Infecção Hospitalar/microbiologia , Enterobacter/enzimologia , Klebsiella pneumoniae/enzimologia , Oxirredutases O-Desmetilantes/genética , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/enzimologia , Acinetobacter baumannii/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Codeína/metabolismo , Enterobacter/genética , Humanos , Klebsiella pneumoniae/genética , Derivados da Morfina/metabolismo , Alcaloides Opiáceos/administração & dosagem , Pseudomonas aeruginosa/genética , Alinhamento de Sequência , Staphylococcus aureus/genética , Tebaína/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-30970111

RESUMO

Multidrug resistance (MDR), virulence and transferable elements potentiate Pseudomonas aeruginosa's role as an opportunistic pathogen creating a high risk for public health. In this study, we evaluated the possible association of multidrug resistance, virulence factors and integrons with intrahospital P. aeruginosa strains isolated from patients at Cumana hospital, Venezuela. Relevant clinical-epidemiological data were collected to study 176 strains (2009-2016) isolated from different hospital units. Bacterial resistance was classified as susceptible, low-level resistant (LDR), multidrug resistant (MDR) and extensively drug-resistant (XDR). Most strains produced pyoverdine, DNase, gelatinase and hemolysin. Around 73% of the strains showed some type of movement. MDR and XDR strains increased from 2009 (24.2% and 4.8%, respectively) to 2016 (53.1% and 18.8%); while LDR decreased from 64.5% to 6.3%. The exoU and exoS genes were found in a significant number of strains (38.1 and 7.4%, respectively). Class I integrons were detected in 35.8% of the strains and the frequency was associated with resistance (42.9, 22.4, 41.4 and 61.9%, for susceptible, LDR, MDR and XDR, respectively). The MDR/XDR strains were positively associated with hemolysins and exoU, but negatively associated with bacterial twitching. MDR/XDR phenotypes were also associated with the Intensive Care Unit (ICU), septicemia, bronchial infection and diabetic foot ulcers, as well as long hospital stay (≥10 days) and previous antimicrobial treatment. High frequency of MDR/XDR strains and their association with class I integrons and virulence factors can increase the infection potential, as well as morbidity and mortality of patients attending this hospital and could spread infection to the community, creating a health risk for the region.


Assuntos
Antibacterianos/farmacologia , Integrons/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Fatores de Virulência/genética , Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana Múltipla , Hospitais Universitários , Humanos , Unidades de Terapia Intensiva , Testes de Sensibilidade Microbiana , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Venezuela , Virulência
11.
Molecules ; 24(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970590

RESUMO

Seven new coralmycin derivatives, coralmycins C (1), D (2), E (3), F (4), G (5), H (6), and I (7), along with three known compounds, cystobactamids 891-2 (8), 905-2 (9), and 507 (10), were isolated from a large-scale culture of the myxobacteria Corallococcus coralloides M23. The structures of these compounds, including their relative stereochemistries, were elucidated by interpretation of their spectroscopic and CD data. The structure-activity relationships of their antibacterial and DNA gyrase inhibitory activities indicated that the para-nitrobenzoic acid unit is critical for the inhibition of DNA gyrase and bacterial growth, while the nitro moiety of the para-nitrobenzoic acid unit and the isopropyl chain at C-4 could be important for permeability into certain Gram-negative bacteria, including Pseudomonas aeruginosa and Klebsiella pneumoniae, and the ß-methoxyasparagine moiety could affect cellular uptake into all tested bacteria. These results could facilitate the chemical optimization of coralmycins for the treatment of multidrug-resistant Gram-negative bacteria.


Assuntos
Antibacterianos , Proteínas de Bactérias/antagonistas & inibidores , DNA Girase , Depsipeptídeos , Klebsiella pneumoniae , Myxococcales/química , Pseudomonas aeruginosa , Inibidores da Topoisomerase II , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia
12.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 4): 270-277, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30950828

RESUMO

The human membrane-bound α/ß-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence of n-octyl ß-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.


Assuntos
Esterases/química , Esterases/isolamento & purificação , Monoacilglicerol Lipases/química , Pseudomonas aeruginosa/enzimologia , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Cristalização , Estabilidade Enzimática , Humanos , Cinética , Especificidade por Substrato , Temperatura Ambiente
13.
Eur J Med Chem ; 171: 209-220, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30925337

RESUMO

The enzyme FabH catalyzes the initial step of fatty acid biosynthesis that is essential for bacterial survival. Therefore, FabH has been identified as an attractive target for the development of new antibacterial agents. We present here the discovery of a promising new series of Pyrazol-Benzimidazole amides with low toxicity and potent FabH inhibitory. Twenty-seven novel compounds have been synthesized, and all the compounds were characterized by 1H NMR, 13C NMR and MS. Afterwards they were evaluated for in-vitro antibacterial activities against E. coli, P. aeruginosa, B. subtilis and S. aureus, along with E. coli FabH inhibition and cytotoxicity test. Some compounds proved to be of low toxicity and potent, especially compound 31 exhibited the most potential to be a new drug with MIC of 0.49-0.98 µg/mL against the tested bacterial strains and IC50 of 1.22 µM against E. coli FabH. Eight analogues 16, 28, 30, 31, 33, 34, 35 and 36 with low range MIC against wild type Xanthomonas Campestris exhibited no inhibition against FabH-deficient mutant strain, which firmly proved the class of compounds arrived at antibacterial activity via interacting with FabH. In silico ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) evaluation also pointed out that these compounds are potential for druggability. Further, effective overall docking scores of all the compounds have been recorded, and docking simulation of compound 31 into E. coli FabH binding pocket has been conducted, where solid binding interactions has been identified.


Assuntos
Bacillus subtilis/enzimologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Ácido Graxo Sintase Tipo II/antagonistas & inibidores , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/enzimologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade
14.
Acta Biochim Pol ; 66(1): 91-100, 2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30831574

RESUMO

Thermally induced unfolding and renaturation capability of alkaline proteases (AprA) of three Pseudomonas aeruginosa strains, i.e. ATCC 27853 and two clinical isolates, was examined. Sequence analyses demonstrated a high level of aprA genes identity (99.24-99.8%) in these bacterial strains. The proteases retained 45-60% and 15% of their activity after pre-treatment at 60oC and 80oC, respectively, whereas pre-incubation at 90-95oC resulted in a higher level of activity than at 80oC. Zymography analyses and immunoblotting with AprA antiserum suggested a high thermostability and renaturation capability of the studied enzymes in comparison to another P. aeruginosa protease, elastase B. An intrinsic capability of renaturation of P. aeruginosa AprA was confirmed by fluorescence spectra of the native, thermally denatured, and renatured enzyme. The value of the fluorescence intensity of the denatured and subsequently cooled enzyme recovered to about 80% of the value of the native protein fluorescence intensity. Moreover, pre-incubation of the enzyme at 60oC and 90oC exerted only a slight effect on the intensity of absorbance and the shape of the amide I band, as demonstrated by Fourier transform infrared (FTIR) spectroscopy performed after subsequent cooling of the pre-treated enzyme. The results indicated a high renaturation capability of the P. aeruginosa AprA proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Pseudomonas aeruginosa/enzimologia , Espectroscopia de Infravermelho com Transformada de Fourier
15.
J Biosci ; 44(1)2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30837358

RESUMO

A 48 kDa ZuhP13 elastase from P. aeruginosa isolated from a urine sample was successfully purified to 8.8-fold and 39% recovery by DEAE-Sepharose CL-6B and Sephadex G-100 chromatography. Its ideal reaction values were pH 7.5 and 40°C. It showed stability at pH 6-9 for 1 h and up to 60°C for 30 min with midpoint temperature (Tm) at 61.3°C and isoelectric value (pI) at 5.6+/-0.2. Its Km and catalytic efficiency (Kcat/Km) for the substrate azocasein were 1.3 mg/mL and 4.629107 M-1s-1, respectively. On contrary to most P. aeruginosa proteases, Zn2+, EDTA, 2,2'-bipyridine and o-phenanthroline showed slight inhibition upon its activity, while, the elastase inhibitors (elastatinal and elastase inhibitor II) and the serine protease inhibitors (TLCK, PMSF, SBTI, and aprotinin) markedly decreased the enzymatic activity. Taken together, we suggest that ZuhP13 is a serine elastase-type. Interestingly, the tested enzyme showed both hemolytic and hemorrhagic activities in vivo. Furthermore, it induced nuclear lysis yielding hyperchromatism within leaky and malformed hepatocytes, suggesting ZuhP13 elastase as a high molecular weight potential pathological agent.


Assuntos
Elastase Pancreática/isolamento & purificação , Elastase Pancreática/farmacologia , Pseudomonas aeruginosa/enzimologia , Virulência/genética , Catálise , Estabilidade Enzimática , Hepatócitos/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Elastase Pancreática/química , Elastase Pancreática/genética , Pseudomonas aeruginosa/patogenicidade , Serina/química , Serina/metabolismo , Inibidores de Serino Proteinase/farmacologia
16.
Methods Mol Biol ; 1954: 255-268, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864138

RESUMO

The donor substrates for the biosynthesis of bacterial polysaccharides include UDP-Glc/Gal and UDP-GlcNAc/GalNAc. The conversion of these nucleotide sugars is catalyzed by 4-epimerases. The wbpP gene of Pseudomonas aeruginosa encodes a 4-epimerase that has a preference for UDP-GlcNAc/GalNAc as substrates. Other 4-epimerases have broad specificities or preference for UDP-Glc/Gal. We have developed coupled assays where the 4-epimerase product is used as a donor substrate for glycosyltransferases that are highly specific for the nucleotide sugar structure. We describe here a method for the study of substrate specificity of WbpP, using coupled assays employing four different glycosyltransferases. These protocols can be applied to the identification and characterization of novel 4-epimerases and to determine their substrate specificities.


Assuntos
Ensaios Enzimáticos/métodos , Glicosiltransferases/metabolismo , Pseudomonas aeruginosa/enzimologia , Racemases e Epimerases/metabolismo , Polissacarídeos Bacterianos/metabolismo , Pseudomonas aeruginosa/metabolismo , Especificidade por Substrato
17.
Mol Cell ; 74(1): 132-142.e5, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30872121

RESUMO

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/metabolismo , Mimetismo Molecular , Pseudomonas aeruginosa/enzimologia , RNA Bacteriano/metabolismo , RNA Guia/metabolismo , Proteínas Virais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Guia/química , RNA Guia/genética , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia
18.
Appl Microbiol Biotechnol ; 103(9): 3753-3760, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30919102

RESUMO

Pseudomonas aeruginosa are ubiquitous γ-proteobacteria capable of producing the biosurfactant rhamnolipids (RL) and the polymer polyhydroxyalkanoate (PHA). RL are glycolipids with high biotechnological potential, whereas PHA is used for the production of biodegradable plastics. It has been proposed that the ß-oxidation pathway provides intermediates for RL biosynthesis, even when using a non-fatty acid carbon source for growth, while an intermediate of de novo fatty acid biosynthesis (FASII) pathway [(R)-3-hydroxyacyl-ACP] is used for PHA biosynthesis. The aim of this work is to study the inter-relationship of the RL and PHA biosynthetic pathways in a culture medium with a non-fatty acid carbon source, focusing on the role of FASII and ß-oxidation in supplying the substrates for the first step in RL and PHA synthesis, carried out by the RhlA and PhaG enzymes, respectively. The PHA synthases (PhaC1 and PhaC2) are only able to use CoA-linked 3-hydroxy acids and the PhaG enzyme catalyzes the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA, the substrate of PhaC1 and PhaC2. RhlA in turn catalyzes the synthesis of the RL precursor 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAA) by the dimerization of two 3-hydroxyalkanoic acid molecules (that have been shown to be also (R)-3-hydroxyacyl-ACP). In this work, we show that RhlA can produce both RL and PHA precursors (presumably CoA-linked HAA), that the blockage of carbon flux through ß-oxidation pathway does not decrease RL titer, and that the enoyl-CoA hydratase RhlY and enoyl-CoA hydratase/isomerase RhlZ produce the main fatty acids precursor of RL using as substrate also a FASII intermediate (presumably (S)-3-hydroxyacyl-CoA).


Assuntos
Ácidos Graxos/metabolismo , Glicolipídeos/biossíntese , Poli-Hidroxialcanoatos/biossíntese , Pseudomonas aeruginosa/metabolismo , Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Oxirredução , Pseudomonas aeruginosa/enzimologia
19.
Inorg Chem ; 58(14): 8969-8982, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30788970

RESUMO

Nickel-containing enzymes such as methyl coenzyme M reductase (MCR) and carbon monoxide dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) play a critical role in global energy conversion reactions, with significant contributions to carbon-centered processes. These enzymes are implied to cycle through a series of nickel-based organometallic intermediates during catalysis, though identification of these intermediates remains challenging. In this work, we have developed and characterized a nickel-containing metalloprotein that models the methyl-bound organometallic intermediates proposed in the native enzymes. Using a nickel(I)-substituted azurin mutant, we demonstrate that alkyl binding occurs via nucleophilic addition of methyl iodide as a methyl donor. The paramagnetic NiIII-CH3 species initially generated can be rapidly reduced to a high-spin NiII-CH3 species in the presence of exogenous reducing agent, following a reaction sequence analogous to that proposed for ACS. These two distinct bioorganometallic species have been characterized by optical, EPR, XAS, and MCD spectroscopy, and the overall mechanism describing methyl reactivity with nickel azurin has been quantitatively modeled using global kinetic simulations. A comparison between the nickel azurin protein system and existing ACS model compounds is presented. NiIII-CH3 Az is only the second example of two-electron addition of methyl iodide to a NiI center to give an isolable species and the first to be formed in a biologically relevant system. These results highlight the divergent reactivity of nickel across the two intermediates, with implications for likely reaction mechanisms and catalytically relevant states in the native ACS enzyme.


Assuntos
Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Níquel/química , Compostos Organometálicos/química , Azurina/genética , Azurina/metabolismo , Catálise , Cromatografia Gasosa , Regulação Bacteriana da Expressão Gênica , Cinética , Fenômenos Magnéticos , Mutação , Compostos Organometálicos/metabolismo , Pseudomonas aeruginosa/enzimologia , Análise Espectral
20.
Chem Pharm Bull (Tokyo) ; 67(2): 125-129, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30713272

RESUMO

Broadened antibacterial activity was introduced to rhodanine derivatives targeting Mycobacterial tuberculosis enoyl-acyl carrier protein reductase (Mtb InhA) by recruiting feature of xacins to bring DNA Gyrase B inhibitory capability. This is significant for preventing further bacterial injections in the tuberculosis treatment. The most potent compound Cy14 suggested comparable bioactivity (IC50 = 3.18 µM for Mtb InhA; IC50 = 10 nM for DNA Gyrase B) with positive controls. Structure-activity relationship discussion and molecular docking model revealed the significance of rhodanine moiety and derived methoxyl on meta-position, pointing out orientations for future modification.


Assuntos
Antibacterianos , Rodanina/análogos & derivados , Proteína de Transporte de Acila , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oxirredutases/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Rodanina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Relação Estrutura-Atividade
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