RESUMO
Bacteria use the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) to control biofilm formation and other key phenotypes in response to environmental signals. Changes in oxygen levels can alter c-di-GMP signaling through a family of proteins termed globin coupled sensors (GCS) that contain diguanylate cyclase domains. Previous studies have found that GCS diguanylate cyclase activity is controlled by ligand binding to the heme within the globin domain, with oxygen binding resulting in the greatest increase in catalytic activity. Herein, we present evidence that heme-edge residues control O2-dependent signaling in PccGCS, a GCS protein from Pectobacterium carotovorum, by modulating heme distortion. Using enzyme kinetics, resonance Raman spectroscopy, small angle X-ray scattering, and multi-wavelength analytical ultracentrifugation, we have developed an integrated model of the full-length PccGCS tetramer and have identified conformational changes associated with ligand binding, heme conformation, and cyclase activity. Taken together, these studies provide new insights into the mechanism by which O2 binding modulates activity of diguanylate cyclase-containing GCS proteins.
Assuntos
Proteínas de Bactérias , Heme , Pectobacterium carotovorum , Fósforo-Oxigênio Liases , Fósforo-Oxigênio Liases/metabolismo , Fósforo-Oxigênio Liases/química , Heme/química , Heme/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Pectobacterium carotovorum/enzimologia , Conformação Proteica , Oxigênio/química , Oxigênio/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Proteínas de Escherichia coliRESUMO
Numerous functions in pathogenic Pectobacterium are regulated by quorum sensing (QS). Two different aiiA genes isolated from Bacillus sp. A24(aiiAA24) and Bacillus sp. DMS133(aiiADMS133) were used. Both genes encode acyl-homoserine lactonase (AiiA), which disrupts QS in Pectobacterium. To investigate the effect of different AiiAs on the inhibition of Pectobacterium carotovorum pathogenicity, two aiiA genes from different Bacillus strains were cloned and the resulting plasmids pME6863 (aiiAA24) and pME7080 (aiiADMS133) were transformed into P. carotovorum EMPCC cells. The effects of different lactonases on virulence features such as enzymatic activity, twitching and swimming motilities, and production of pellicle and biofilm formation were investigated. In EMPCC/pME6863, twitching and swimming motilities, and pellicle production were significantly reduced compared with EMPCC/pME7080. Quantitative real-time PCR (qRT-PCR) was used to measure virulence gene expression in transformed cells compared with expression levels in wild-type EMPCC. The expression of peh and hrpL genes was greatly reduced in EMPCC/pME6863 compared with EMPCC/pME7080. The sequence alignment and molecular dynamic modeling of two different AiiAA24 and AiiADMS133 proteins suggested that the replacement of proline 210 from AiiAA24 to serine in AiiADMS133 caused the reduction of enzyme activity in AiiADMS133.
Assuntos
Bacillus , Proteínas de Bactérias , Hidrolases de Éster Carboxílico , Percepção de Quorum , Bacillus/genética , Bacillus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Biofilmes/crescimento & desenvolvimento , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/enzimologia , Pectobacterium carotovorum/patogenicidade , Virulência , Regulação Bacteriana da Expressão Gênica , Clonagem Molecular , MetaloendopeptidasesRESUMO
Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Pectobacterium carotovorum/enzimologia , Poligalacturonase/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Ácidos Hexurônicos/metabolismo , Pectinas/metabolismo , Pectobacterium carotovorum/genética , Poligalacturonase/genética , Cloreto de Potássio/metabolismo , Regiões Promotoras GenéticasRESUMO
The type II secretion system (T2SS) transports fully folded proteins of various functions and structures through the outer membrane of Gram-negative bacteria. The molecular mechanisms of substrate recruitment by T2SS remain elusive but a prevailing view is that the secretion determinants could be of a structural nature. The phytopathogenic γ-proteobacteria, Pectobacterium carotovorum and Dickeya dadantii, secrete similar sets of homologous plant cell wall degrading enzymes, mainly pectinases, by similar T2SSs, called Out. However, the orthologous pectate lyases Pel3 and PelI from these bacteria, which share 67% of sequence identity, are not secreted by the counterpart T2SS of each bacterium, indicating a fine-tuned control of protein recruitment. To identify the related secretion determinants, we first performed a structural characterization and comparison of Pel3 with PelI using X-ray crystallography. Then, to assess the biological relevance of the observed structural variations, we conducted a loop-substitution analysis of Pel3 combined with secretion assays. We showed that there is not one element with a definite secondary structure but several distant and structurally flexible loop regions that are essential for the secretion of Pel3 and that these loop regions act together as a composite secretion signal. Interestingly, depending on the crystal contacts, one of these key secretion determinants undergoes disorder-to-order transitions that could reflect its transient structuration upon the contact with the appropriate T2SS components. We hypothesize that such T2SS-induced structuration of some intrinsically disordered zones of secretion substrates could be part of the recruitment mechanism used by T2SS.
Assuntos
Proteínas de Bactérias/química , Dickeya/enzimologia , Pectobacterium carotovorum/enzimologia , Polissacarídeo-Liases/química , Sistemas de Secreção Tipo II/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Parede Celular/química , Parede Celular/microbiologia , Clonagem Molecular , Cristalografia por Raios X , Dickeya/classificação , Dickeya/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Pectobacterium carotovorum/classificação , Pectobacterium carotovorum/genética , Filogenia , Células Vegetais/química , Células Vegetais/microbiologia , Plantas/química , Plantas/microbiologia , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/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 de Sequência de Aminoácidos , Sistemas de Secreção Tipo II/genética , Sistemas de Secreção Tipo II/metabolismoRESUMO
Complex heterogeneous systems, such as micelles or blood plasma, represent a particularly challenging environment to measure the catalytic parameters of some enzymes, including l-asparaginase. Existing methods are strongly interfered by the presence of plasma proteins, amino acids, as well as other components of plasma. Here we show that FTIR spectroscopy enables continuous real-time measurement of catalytic activity of l-asparaginase, in native and in PEG-chitosan conjugated form, in aqueous solutions as well as in heterogeneous non-transparent multicomponent systems, including colloidal systems or blood plasma, with minimal or no sample preparation. The approach developed is potentially applicable to other enzymatic reactions where the spectroscopic properties of substrate and product do not allow direct measurement with absorption or fluorescence spectroscopy.
Assuntos
Asparaginase/análise , Asparaginase/metabolismo , Biocatálise , Quitosana/química , Humanos , Pectobacterium carotovorum/enzimologia , Polietilenoglicóis/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O2-dependent pathways, the mechanism by which the O2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O2-dependent GCS activity.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella pertussis/enzimologia , Oxigênio/metabolismo , Pectobacterium carotovorum/enzimologia , Fósforo-Oxigênio Liases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Bordetella pertussis/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Cinética , Pectobacterium carotovorum/genética , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/genética , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.
Assuntos
Aminoácidos/análise , Hidrolases de Éster Carboxílico , Pectobacterium carotovorum/enzimologia , Pectobacterium carotovorum/genética , Engenharia de Proteínas , Substituição de Aminoácidos , Brassica rapa/microbiologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Ativação Enzimática/genética , Mutação , Pectobacterium carotovorum/patogenicidade , Virulência/genéticaRESUMO
Acute lymphoblastic leukemia (ALL) is a type of cancer with a high incidence in children. The enzyme l-asparaginase (ASNase) constitutes a key element in the treatment of this disease. Four formulations of ASNase from a bacterial source are currently available. However, these formulations are characterized by their high immunogenicity, resulting in the inactivation of the drug, as well as in the occurrence of hypersensitivity reactions in a large number of patients. In this work, we performed an immunoinformatic analysis in order to clarify structural aspects of the immunogenicity of the asparaginase from Escherichia coli and Erwinia carotovora. For this purpose, we performed the prediction of immunogenic and allergenic epitopes in the structure of asparaginases by using the relative frequency of immunogenic peptides for the eight alleles most frequently distributed worldwide. This study showed that there are no significant differences in the level of immunogenicity between the two enzymes, while asparaginase from E. coli presented a higher relative frequency of allergenic epitopes. These results are consistent with previously published reports. However, from a structural point of view, to the best of our knowledge, this is the first report describing the structural determinants that contribute to the hypersensitivity response to this treatment.
Assuntos
Asparaginase/imunologia , Proteínas de Bactérias/imunologia , Epitopos/imunologia , Escherichia coli/enzimologia , Pectobacterium carotovorum/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Sequência de Aminoácidos , Asparaginase/efeitos adversos , Asparaginase/uso terapêutico , Proteínas de Bactérias/química , Proteínas de Bactérias/uso terapêutico , Criança , Simulação por Computador , Epitopos/química , Escherichia coli/genética , Humanos , Hipersensibilidade/etiologia , Hipersensibilidade/imunologia , Pectobacterium carotovorum/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Enzymatic prospection indicated that L-asparaginase from Erwinia carotovora (ECAR-LANS) posses low glutaminase activity and much effort has been made to produce therapeutic ECAR-LANS. However, its low stability precludes its use in therapy. Herein, biochemical and biophysical assays provided data highlighting the influence of solubilization and storage into ECAR-LANS structure, stability, and activity. Moreover, innovations in recombinant expression and purification guaranteed the purification of functional tetramers. According to solubilization condition, the L-asparaginase activity and temperature of melting ranged up to 25-32%, respectively. CD spectra indicate the tendency of ECAR-LANS to instability and the influence of ß-structures in activity. These results provide relevant information to guide formulations with prolonged action in the bloodstream.
Assuntos
Asparaginase/metabolismo , Pectobacterium carotovorum/enzimologia , Citoplasma/enzimologia , Estabilidade Enzimática , Fluorescência , Periplasma/enzimologiaRESUMO
BACKGROUND: L-ASNase (L-asparagine aminohydrolase EC 3.5.1.1) is used for the conversion of L-asparagine to L-aspartic acid and ammonia and also it was found as an agent of chemotherapeutic property according to recent patents. It is known as an anti-cancer agent and recently it has received an immense attention. Various microorganisms have the ability to secrete the L-ASNase. It is famous world-wide as anti-tumor medicine for acute lymphoblastic leukemia and lymphosarcoma. L-ASNase helps in deamination of Asparagine and Glutamine. SOURCE: L-ASNase mainly found in two bacterial sources; Escherichia coli and Erwinia carotovora. Isolation from plants: Endophytes were also a great source of L-ASNase. It was isolated from four types of plants named as; C. citratus, O. diffusa, M. koengii, and also P. bleo. APPLICATIONS: L-ASNase is used as a potential anti-tumor medicine. It plays a very much essential role for the growth of tumor cells. Tumor cells require a lot of asparagine for their growth. But ASNase converts to aspartate and ammonia from asparagine. So the tumor cell does not proliferate and fails to survive. The L-ASNase is used as the medicine for the major type of cancer like acute lymphocytic leukemia (ALL), brain. It also used as a medicine for central nervous system (CNS) tumors, and also for neuroblastoma. Two types of L-ASNase have been found. CONCLUSION: L-ASNase becomes a powerful anti-tumor medicine and researchers should develop a potent strain of asparaginase which can produce asparaginase in the industrial level. It is also used in the pharmaceutical industry and food industry on a broader scale.
Assuntos
Antineoplásicos/química , Asparaginase/química , Asparaginase/isolamento & purificação , Patentes como Assunto , Antineoplásicos/uso terapêutico , Asparaginase/genética , Asparagina/química , Escherichia coli/enzimologia , Glutamina/química , Humanos , Neuroblastoma/tratamento farmacológico , Pectobacterium carotovorum/enzimologiaRESUMO
Hfq is a RNA chaperone and participates in a wide range of cellular processes and pathways. In this study, mutation of hfq gene from Pectobacterium carotovorum subsp. carotovorum PccS1 led to significantly reduced virulence and plant cell wall-degrading enzyme (PCWDE) activities. In addition, the mutant exhibited decreased biofilm formation and motility and greatly attenuated carbapenem production as well as secretion of hemolysin coregulated protein (Hcp) as compared with wild-type strain PccS1. Moreover, a higher level of callose deposition was induced in Nicotiana benthamiana leaves when infiltrated with the mutant. A total of 26 small (s)RNA deletion mutants were obtained among a predicted 27 sRNAs, and three mutants exhibited reduced virulence in the host plant. These results suggest that hfq plays a key role in Pectobacterium virulence by positively impacting PCWDE production, secretion of the type VI secretion system, bacterial competition, and suppression of host plant responses.
Assuntos
Biofilmes/crescimento & desenvolvimento , Calla (Planta)/microbiologia , Fator Proteico 1 do Hospedeiro/metabolismo , Pectobacterium carotovorum/enzimologia , Doenças das Plantas/microbiologia , Sistemas de Secreção Tipo VI/metabolismo , Sequência de Aminoácidos , Calla (Planta)/imunologia , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucanos/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/patogenicidade , Pectobacterium carotovorum/fisiologia , Doenças das Plantas/imunologia , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Alinhamento de Sequência , Sistemas de Secreção Tipo VI/genética , VirulênciaRESUMO
Targeting of recombinant proteins to the Escherichia coli periplasm is a desirable industrial processing tool to allow formation of disulphide bonds, aid folding and simplify recovery. Proteins are targeted across the inner membrane to the periplasm by an N-terminal signal peptide. The sequence of the signal peptide determines its functionality, but there is no method to predict signal peptide function for specific recombinant proteins, so multiple signal peptides must be screened for their ability to translocate each recombinant protein, limiting throughput. We present a screening system for optimising signal peptides for translocation of a single chain variable (scFv) antibody fragment employing TEM1 ß-lactamase (Bla) as a C-terminal reporter of periplasmic localisation. The Pectobacterium carotovorum PelB signal peptide was selected as the starting point for a mutagenic screen. ß-lactamase was fused to the C-terminal of scFv and ß-lactamase activity was correlated against scFv translocation. Signal peptide libraries were generated and screened for ß-lactamase activity, which correlated well to scFv::Bla production, although only some high activity clones had improved periplasmic translocation of scFv::Bla. Selected signal peptides were investigated in fed-batch fermentations for production and translocation of scFv::Bla and scFv without the Bla fusion. Improved signal peptides increased periplasmic scFv activity by ~40%.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Testes Genéticos/métodos , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , beta-Lactamases/análise , Genes Reporter , Engenharia Metabólica/métodos , Mutagênese , Pectobacterium carotovorum/enzimologia , Pectobacterium carotovorum/genética , Periplasma/metabolismo , Polissacarídeo-Liases/genética , Transporte Proteico , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , beta-Lactamases/genéticaRESUMO
To prevent the postharvest disease of Chinese cabbage and lettuce, hexanal was used as a control measure to inhibit N-acyl homoserine lactone (AHL) production and extracellular enzymes regulated by quorum-sensing (QS) in their main spoilage strains of Erwinia carotovora and Pseudomonas fluorescens. Firstly, the QS inhibition of hexanal was verified by significantly inhibiting violacein production (pâ¯<â¯0.05) in Chromobacterium violaceum CV026 at sub-MICs. ß-Galactosidase activities which reflected AHL production, were significantly inhibited by hexanal, its inhibitory effect was concentration-dependent under minimal inhibitory concentration (MIC) (pâ¯<â¯0.05). The detected extracellular enzymes activities decreased with the increase of hexanal concentration (pâ¯<â¯0.05), including cellulase, xylanase, pectate lyase, polygalacturonase, and protease. Chinese cabbage soft rot and lettuce leaf scorch could be significantly inhibited by hexanal (pâ¯<â¯0.05) without any phytotoxicity effect, the 1/2 MIC of hexanal showed the best inhibitory effect. And all the above effects showed a dose-dependent. A novel preservation technique in reducing the loss of vegetables due to spoilage based on the QS inhibitor was developed.
Assuntos
Aldeídos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Pectobacterium carotovorum/enzimologia , Pseudomonas fluorescens/enzimologia , Percepção de Quorum/efeitos dos fármacos , Verduras/microbiologia , 4-Butirolactona/análogos & derivados , Chromobacterium/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Conservação de Alimentos/métodos , Glicosídeo Hidrolases/metabolismo , Testes de Sensibilidade Microbiana , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Folhas de Planta , Polissacarídeo-Liases , Verduras/crescimento & desenvolvimentoRESUMO
Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526â U gprot-1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32â h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0 ) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1 ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.
Assuntos
Nitrilas/metabolismo , Oxirredutases/metabolismo , Pectobacterium carotovorum/enzimologia , Sítios de Ligação , Domínio Catalítico , Evolução Molecular Direcionada , Estabilidade Enzimática , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Pectobacterium carotovorum/química , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Mutação Puntual , Engenharia de Proteínas , Pirimidinas/metabolismo , Pirróis/metabolismo , Especificidade por SubstratoRESUMO
The screening of a metagenomic library of 250,000 clones generated from a hypersaline soil (Spain) allowed us to identify a single positive clone which confers the ability to degrade N-acyl homoserine lactones (AHLs). The sequencing of the fosmid revealed a 42,318 bp environmental insert characterized by 46 ORFs. The subcloning of these ORFs demonstrated that a single gene (hqiA) allowed AHL degradation. Enzymatic analysis using purified HqiA and HPLC/MS revealed that this protein has lactonase activity on a broad range of AHLs. The introduction of hqiA in the plant pathogen Pectobacterium carotovorum efficiently interfered with both the synthesis of AHLs and quorum-sensing regulated functions, such as swarming motility and the production of maceration enzymes. Bioinformatic analyses highlighted that HqiA showed no sequence homology with the known prototypic AHL lactonases or acylases, thus expanding the AHL-degrading enzymes with a new family related to the cysteine hydrolase (CHase) group. The complete sequence analysis of the fosmid showed that 31 ORFs out of the 46 identified were related to Deltaproteobacteria, whilst many intercalated ORFs presented high homology with other taxa. In this sense, hqiA appeared to be assigned to the Hyphomonas genus (Alphaproteobacteria), suggesting that horizontal gene transfer had occurred.
Assuntos
Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Deltaproteobacteria/isolamento & purificação , Pectobacterium carotovorum/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Deltaproteobacteria/enzimologia , Deltaproteobacteria/genética , Transferência Genética Horizontal , Espectrometria de Massas , Metagenômica/métodos , Família Multigênica , Fases de Leitura Aberta , Pectobacterium carotovorum/enzimologia , Pectobacterium carotovorum/genética , Percepção de Quorum , Análise de Sequência de DNA , Microbiologia do Solo , EspanhaRESUMO
The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrÐE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrÐ+N17, D60K, F61L and RrÐ+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.
Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Proteínas de Bactérias/farmacologia , Mutação Puntual , Rhodospirillum rubrum/enzimologia , Telomerase/antagonistas & inibidores , Telômero/efeitos dos fármacos , Sequência de Aminoácidos , Antineoplásicos/química , Antineoplásicos/metabolismo , Asparaginase/química , Asparaginase/genética , Asparaginase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Células HL-60 , Humanos , Células Jurkat , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pectobacterium carotovorum/química , Pectobacterium carotovorum/enzimologia , Pectobacterium carotovorum/genética , Plasmídeos/química , Plasmídeos/metabolismo , Estrutura Secundária de Proteína , Rhodospirillum rubrum/química , Rhodospirillum rubrum/genética , Especificidade da Espécie , Relação Estrutura-Atividade , Telomerase/genética , Telomerase/metabolismo , Telômero/química , Wolinella/química , Wolinella/enzimologia , Wolinella/genéticaRESUMO
The type II secretion system (T2SS), which transports selected periplasmic proteins across the outer membrane, has rarely been studied in nonpathogens or in organisms classified as Betaproteobacteria. Therefore, we studied Cupriavidus metallidurans (Cme), a facultative chemilithoautotroph. Gel analysis of extracellular proteins revealed no remarkable differences between the wild type and the T2SS mutants. However, enzyme assays revealed that native extracellular alkaline phosphatase is a T2SS substrate, because activity was 10-fold greater for the wild type than a T2SS mutant. In Cme engineered to produce three Ralstonia solanacearum (Rso) exoenzymes, at least 95% of their total activities were extracellular, but unexpectedly high percentages of these exoenzymes remained extracellular in T2SS mutants cultured in rich broth. These conditions appear to permit an alternative secretion process, because neither cell lysis nor periplasmic leakage was observed when Cme produced a Pectobacterium carotovorum exoenzyme, and wild-type Cme cultured in minimal medium secreted 98% of Rso polygalacturonase, but 92% of this exoenzyme remained intracellular in T2SS mutants. We concluded that Cme has a functional T2SS despite lacking any abundant native T2SS substrates. The efficient secretion of three foreign exoenzymes by Cme is remarkable, but so too is the indication of an alternative secretion process in rich culture conditions. When not transiting the T2SS, we suggest that Rso exoenzymes are probably selectively packaged into outer membrane vesicles. Phylogenetic analysis of T2SS proteins supports the existence of at least three T2SS subfamilies, and we propose that Cme, as a representative of the Betaproteobacteria, could become a new useful model system for studying T2SS substrate specificity.
Assuntos
Proteínas de Bactérias/metabolismo , Cupriavidus/enzimologia , Cupriavidus/metabolismo , Sistemas de Secreção Tipo II/metabolismo , Sistemas de Secreção Tipo II/fisiologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Transporte Biológico , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Celulase/genética , Celulase/metabolismo , Cupriavidus/genética , DNA Bacteriano , Ensaios Enzimáticos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Família Multigênica/genética , Mutação , Pectobacterium carotovorum/enzimologia , Filogenia , Poligalacturonase/genética , Poligalacturonase/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Sistemas de Translocação de Proteínas/classificação , Sistemas de Translocação de Proteínas/genética , Sistemas de Translocação de Proteínas/metabolismo , Sistemas de Translocação de Proteínas/fisiologia , Ralstonia solanacearum/enzimologia , Alinhamento de Sequência , Sistemas de Secreção Tipo II/classificação , Sistemas de Secreção Tipo II/genéticaRESUMO
The effect of dissolved oxygen (DO) level and pH (controlled/uncontrolled) was first studied to enhance the production of novel glutaminase-free L-asparaginase by Pectobacterium carotovorum MTCC 1428 in a batch bioreactor. The optimum level of DO was found to be 20%. The production of L-asparaginase was found to be maximum when pH of the medium was maintained at 8.5 after 12 h of fermentation. Under these conditions, P. carotovorum produced 17.97 U/mL of L-asparaginase corresponding to the productivity of 1497.50 U/L/h. The production of L-asparaginase was studied in fed-batch bioreactor by feeding L-asparagine (essential substrate for production) and/or glucose (carbon source for growth) at the end of the reaction period of 12 h. The initial medium containing both L-asparagine and glucose in the batch mode and L-asparagine in the feeding stream was found to be the best combination for enhanced production of glutaminase-free L-asparaginase. Under this condition, the L-asparaginase production was increased to 38.8 U/mL, which corresponded to a productivity of 1615.8 U/L/h. The production and productivity were increased by 115.8% and 7.9%, respectively, both of which are higher than those obtained in the batch bioreactor experiments.
Assuntos
Asparaginase/biossíntese , Reatores Biológicos , Pectobacterium carotovorum/enzimologia , Asparaginase/metabolismo , Meios de Cultura , Glutaminase/metabolismoRESUMO
N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.
Assuntos
Antineoplásicos Fitogênicos/química , Asparaginase/química , Proteínas de Bactérias/química , Pectobacterium carotovorum/química , Polietilenoglicóis/química , Antineoplásicos Fitogênicos/biossíntese , Antineoplásicos Fitogênicos/farmacologia , Asparaginase/biossíntese , Asparaginase/genética , Asparaginase/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Gel , Clonagem Molecular , Reagentes de Ligações Cruzadas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HL-60 , Humanos , Células Jurkat , Células K562 , Pectobacterium carotovorum/enzimologia , Polietilenoglicóis/farmacologia , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Succinimidas/químicaRESUMO
Bacteria sense their environment to alter phenotypes, including biofilm formation, to survive changing conditions. Heme proteins play important roles in sensing the bacterial gaseous environment and controlling the switch between motile and sessile (biofilm) states. Globin coupled sensors (GCS), a family of heme proteins consisting of a globin domain linked by a central domain to an output domain, are often found with diguanylate cyclase output domains that synthesize c-di-GMP, a major regulator of biofilm formation. Characterization of diguanylate cyclase-containing GCS proteins from Bordetella pertussis and Pectobacterium carotovorum demonstrated that cyclase activity is controlled by ligand binding to the heme within the globin domain. Both O2 binding to the heme within the globin domain and c-di-GMP binding to a product-binding inhibitory site (I-site) within the cyclase domain control oligomerization states of the enzymes. Changes in oligomerization state caused by c-di-GMP binding to the I-site also affect O2 kinetics within the globin domain, suggesting that shifting the oligomer equilibrium leads to broad rearrangements throughout the protein. In addition, mutations within the I-site that eliminate product inhibition result in changes to the accessible oligomerization states and decreased catalytic activity. These studies provide insight into the mechanism by which ligand binding to the heme and I-site controls activity of GCS proteins and suggests a role for oligomerization-dependent activity in vivo.