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1.
Nat Commun ; 12(1): 4466, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294725

RESUMO

Macrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.


Assuntos
Antibacterianos/farmacologia , Cetolídeos/farmacologia , Macrolídeos/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Antibacterianos/química , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Resistência Microbiana a Medicamentos/genética , Eritromicina/química , Eritromicina/farmacologia , Genes Bacterianos , Cetolídeos/química , Cetolídeos/farmacocinética , Macrolídeos/química , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Insercional , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , Ribossomos/efeitos dos fármacos
2.
Elife ; 102021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34328418

RESUMO

In Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labeling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labeling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The 'closed' octamer interconverts with other oligomers in a phosphorylation-dependent manner and, unlike these 'open' forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fosforilação , Proteínas Quinases/química
3.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074047

RESUMO

In this review, we chart the major milestones in the research progress on the DyP-type peroxidase family over the past decade. Though mainly distributed among bacteria and fungi, this family actually exhibits more widespread diversity. Advanced tertiary structural analyses have revealed common and different features among members of this family. Notably, the catalytic cycle for the peroxidase activity of DyP-type peroxidases appears to be different from that of other ubiquitous heme peroxidases. DyP-type peroxidases have also been reported to possess activities in addition to peroxidase function, including hydrolase or oxidase activity. They also show various cellular distributions, functioning not only inside cells but also outside of cells. Some are also cargo proteins of encapsulin. Unique, noteworthy functions include a key role in life-cycle switching in Streptomyces and the operation of an iron transport system in Staphylococcus aureus, Bacillus subtilis and Escherichia coli. We also present several probable physiological roles of DyP-type peroxidases that reflect the widespread distribution and function of these enzymes. Lignin degradation is the most common function attributed to DyP-type peroxidases, but their activity is not high compared with that of standard lignin-degrading enzymes. From an environmental standpoint, degradation of natural antifungal anthraquinone compounds is a specific focus of DyP-type peroxidase research. Considered in its totality, the DyP-type peroxidase family offers a rich source of diverse and attractive materials for research scientists.


Assuntos
Antraquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Lignina/metabolismo , Peroxidases/química , Peroxidases/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Catálise , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Fungos/crescimento & desenvolvimento , Ferro/metabolismo , Oxirredução , Filogenia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo , Streptomyces/enzimologia , Streptomyces/metabolismo
4.
Nucleic Acids Res ; 49(12): 7088-7102, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34157109

RESUMO

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase-α, ß, ß'. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the ß or ß' subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Endorribonucleases/fisiologia , RNA Mensageiro/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Endorribonucleases/genética , Evolução Molecular , Deleção de Genes , Duplicação Gênica , Genes Bacterianos , Homeostase , Mutação , Supressão Genética , Transcrição Genética , Transcriptoma
5.
Int J Biol Macromol ; 182: 1590-1601, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34015407

RESUMO

Pancreatic cancer is the fourth most lethal cancer type worldwide. Due to multiple levan applications including anticancer activities, studies related to levansucrase production are of interest. To our knowledge, levan effect on pancreatic cancer cells has not been tested previously. In this work, among eighteen bacterial honey isolates, Bacillus subtilis MT453867 showed the highest levan yield (33 g/L) and levansucrase production (8.31 U/mL). One-factor-at-a-time technique increased levansucrase activity by 60% when MgSO4 was eliminated. The addition of 60 g/L banana peels enhanced the enzyme activity (192 U/mL). Placket Burman design determined the media composition for maximum levan yield (54.8 g/L) and levansucrase production (505 U/mL). The identification of levan was confirmed by thin-layer chromatography, Fourier-Transform Infrared spectrometric analysis, 13C-nuclear-magnetic resonance, and 1H-nuclear-magnetic resonance. Both crude and dialyzed levan completely inhibited the pancreatic cancer cell line at 100 ppm with no cytotoxicity on the normal retinal cell line. The LD50 of crude levan was 4833 mg/kg body weight. Levan had strong antioxidant activity and significantly reduced the expression of CXCR4 and MCM7 genes in pancreatic cancer cells with significant DNA fragmentation. In conclusion, Bacillus subtilis MT453867 levan is a promising adjunct to pancreatic-anticancer agents with both anti-cancer and chemoprotective effects.


Assuntos
Antineoplásicos/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Frutanos/metabolismo , Hexosiltransferases/metabolismo , Antineoplásicos/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Frutanos/farmacologia , Humanos , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores CXCR4/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
6.
Chem Commun (Camb) ; 57(36): 4460-4463, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33949502

RESUMO

We report a facile and reversible method to immobilize a broad range of His6-tagged proteins on the E. coli cell surface through Fe(iii)-metal complexes. A His6-tagged eGFP and four His6-tagged enzymes were successfully immobilized on the cell surface. Additionally, a hydrogel sheath around E. coli cells was generated by immobilized His6-tagged HRP.


Assuntos
Oxirredutases do Álcool/metabolismo , Escherichia coli/metabolismo , Compostos Férricos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Lacase/metabolismo , Lipase/metabolismo , Oxirredutases do Álcool/química , Bacillus licheniformis/enzimologia , Bacillus subtilis/enzimologia , Candida tropicalis/enzimologia , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/química , Escherichia coli/citologia , Compostos Férricos/química , Proteínas de Fluorescência Verde/química , Histidina/química , Histidina/metabolismo , Lacase/química , Lipase/química , Oligopeptídeos/química , Oligopeptídeos/metabolismo
7.
Commun Biol ; 4(1): 468, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850233

RESUMO

Microbes commonly display great genetic plasticity, which has allowed them to colonize all ecological niches on Earth. Bacillus subtilis is a soil-dwelling organism that can be isolated from a wide variety of environments. An interesting characteristic of this bacterium is its ability to form biofilms that display complex heterogeneity: individual, clonal cells develop diverse phenotypes in response to different environmental conditions within the biofilm. Here, we scrutinized the impact that the number and variety of the Rap-Phr family of regulators and cell-cell communication modules of B. subtilis has on genetic adaptation and evolution. We examine how the Rap family of phosphatase regulators impacts sporulation in diverse niches using a library of single and double rap-phr mutants in competition under 4 distinct growth conditions. Using specific DNA barcodes and whole-genome sequencing, population dynamics were followed, revealing the impact of individual Rap phosphatases and arising mutations on the adaptability of B. subtilis.


Assuntos
Adaptação Fisiológica/genética , Bacillus subtilis/fisiologia , Genes Bacterianos , Família Multigênica , Monoéster Fosfórico Hidrolases/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum
8.
Phys Chem Chem Phys ; 23(12): 7302-7312, 2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33876090

RESUMO

Solvent-free enzymes hold the promise of being able to deliver higher activity at elevated temperatures by virtue of them being not limited by the boiling point of the solvent. They have been realized in the liquid phase through a polymer surfactant coating on the protein surface. However, a clear understanding of intermolecular interactions, structure, dynamics, and the behaviour of the minuscule amount of water present in the solvent-free protein liquid is essential to enhance the activity of these biofluids. Using atomistic molecular dynamics simulations, we demonstrate that the scaled spatial correlations between proteins in the hybrid liquid phase of Lipase A enzymes are comparable to the inter-particle correlations in a noble gas fluid. The hydrophilic region of the surfactants forms a coronal layer around each enzyme which percolates throughout the liquid, while the hydrophobic parts are present as disjointed clusters. Inter-surfactant interactions, determined to be attractive and in the range of -200 to -300 kcal mol-1, stabilize the liquid state. While the protein retains its native state conformational dynamics in the solvent-free form, the fluxionality of its side chains is much reduced; at 333 K, the latter is found to be equivalent to that of the enzyme in an aqueous solution at 249 K. Despite the sluggishness of the solvent-free enzyme, some water molecules exhibit high mobility and transit between enzymes primarily via the interspersed hydrophilic regions. These microscopic insights offer ideas to improve substrate diffusion in the liquid to enable the enhancement of catalytic activity.


Assuntos
Lipase/química , Simulação de Dinâmica Molecular , Tensoativos/química , Bacillus subtilis/enzimologia , Interações Hidrofóbicas e Hidrofílicas , Lipase/metabolismo , Estrutura Molecular , Tensoativos/metabolismo
9.
mBio ; 12(2)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824210

RESUMO

The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few dehydrogenases in central carbon metabolism that reduce NADP+ with electrons derived from the catabolism of nutrients. In the case of Bacillus subtilis growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in B. subtilis that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative 13C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing in vivo and demonstrated relevant NADPH-oxidizing activity by YtsJ in vitro To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in Escherichia coli MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance.IMPORTANCE A new mechanism for NADPH balancing was discovered in Bacillus subtilis It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP+ in response to instantaneous needs.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Malato Desidrogenase/metabolismo , NADP/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Glucose/metabolismo , Malato Desidrogenase/genética , Malatos/metabolismo , Oxirredução
10.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810099

RESUMO

The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence.


Assuntos
Bacillus subtilis/enzimologia , Histidina Quinase/química , Multimerização Proteica , Algoritmos , Estabilidade Enzimática , Histidina Quinase/metabolismo , Concentração de Íons de Hidrogênio , Modelos Químicos , Modelos Moleculares , Fosforilação , Conformação Proteica , Desnaturação Proteica , Análise Espectral , Relação Estrutura-Atividade , Temperatura
11.
J Biosci Bioeng ; 131(6): 605-612, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33814275

RESUMO

The structures of Aspergillus oryzae α-amylase were determined in a tetragonal crystal, having one molecule as asymmetric unit, and a monoclinic crystal with two molecules as asymmetric unit. Both crystal forms were obtained from trace contaminants of an old commercial lipase preparation. Structures were determined and refined to 1.65 Å and 1.43 Å resolution respectively. The latter crystal has a non-crystallographic (NCS) twofold axis within the asymmetric unit. Glycosylation at Asn197 is evident, and in the tetragonal crystal can be seen to include three, partially disordered sugar residues following the initial N-acetyl glucosamine (NAG). Superposition of the tetragonal crystal model on the α-amylases from Bacillus subtilis (PDB:1BAG), pig pancreas (PDB:3L2L), and barley (PDB:1AMY), show a high degree of coincidence, particularly for the (ß/α)8-barrel domains, and especially within the active site. Using this structural agreement between amylases, we extrapolated the binding model of a six residue, limit dextrin found in pig pancreas α-amylase to the A. oryzae enzyme model, which predicts substrate interacting amino acid residues.


Assuntos
Aspergillus oryzae/enzimologia , alfa-Amilases/química , Amilases/metabolismo , Animais , Aspergillus oryzae/metabolismo , Bacillus subtilis/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Hordeum/enzimologia , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , alfa-Amilases Pancreáticas/química , Conformação Proteica , Estrutura Terciária de Proteína , Suínos/metabolismo , alfa-Amilases/metabolismo
12.
Molecules ; 26(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806149

RESUMO

Bacillus subtilis SH21 was observed to produce an antifungal protein that inhibited the growth of F. solani. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC-MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of F. solani by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against F. solani was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of Streptomyces sp. N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.


Assuntos
Antifúngicos , Bacillus subtilis/enzimologia , Proteínas de Bactérias , Fusarium/crescimento & desenvolvimento , Glicosídeo Hidrolases , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/farmacologia
13.
Molecules ; 26(6)2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808840

RESUMO

α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, BsAbfA, was cloned from Bacilus subtilis, and its codons were optimized for efficient expression in E. coli BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Site-directed mutagenesis of BsAbfA was performed to verify the catalytic site, and the molecular mechanism of BsAbfA catalyzing ginsenoside Rc was analyzed by molecular docking, using the homology model of sequence alignment with other ß-glycosidases. The results show that the purified BsAbfA had a specific activity of 32.6 U/mg. Under optimal conditions (pH 5, 40 °C), the kinetic parameters Km of BsAbfA for pNP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the Kcat/Km were 181.5 s-1 mM-1 and 197.8 s-1 mM-1, respectively. More than 90% of ginsenoside Rc could be transformed by 12 U/mL purified BsAbfA at 40 °C and pH 5 in 24 h. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA are important in recognizing ginsenoside Rc effectively, and to make it enter the active pocket to hydrolyze the outer arabinofuranosyl moieties at C20 position. These remarkable properties and the catalytic mechanism of BsAbfA provide a good alternative for the effective biotransformation of the major ginsenoside Rc into Rd.


Assuntos
Substituição de Aminoácidos , Bacillus subtilis , Proteínas de Bactérias , Ginsenosídeos/química , Glicosídeo Hidrolases , Mutagênese Sítio-Dirigida , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Mutação de Sentido Incorreto , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
14.
J Biol Chem ; 296: 100519, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33684445

RESUMO

Endo-ß-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the ß-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-ß-N-acetylmuramidases, which catalyze an exo-lytic cleavage of ß-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-ß-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl ß-MurNAc and the peptidoglycan-derived disaccharide MurNAc-ß-1,4-GlcNAc. Together with the exo-ß-N-acetylglucosaminidase NagZ and the exo-muramoyl-l-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www.cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.


Assuntos
Acetilglucosamina/metabolismo , Bacillus subtilis/enzimologia , Glicosídeo Hidrolases/metabolismo , Ácidos Murâmicos/metabolismo , Peptidoglicano/metabolismo , Cristalografia por Raios X , Glicosídeo Hidrolases/química , Hidrólise , Conformação Proteica
15.
J Biol Chem ; 296: 100518, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33684446

RESUMO

Reversible phosphorylation relies on highly regulated kinases and phosphatases that target specific substrates to control diverse cellular processes. Here, we address how protein phosphatase activity is directed to the correct substrates under the correct conditions. The serine/threonine phosphatase SpoIIE from Bacillus subtilis, a member of the widespread protein phosphatase 2C (PP2C) family of phosphatases, is activated by movement of a conserved α-helical element in the phosphatase domain to create the binding site for the metal cofactor. We hypothesized that this conformational switch could provide a general mechanism for control of diverse members of the PP2C family of phosphatases. The B. subtilis phosphatase RsbU responds to different signals, acts on a different substrates, and produces a more graded response than SpoIIE. Using an unbiased genetic screen, we isolated mutants in the α-helical switch region of RsbU that are constitutively active, indicating conservation of the switch mechanism. Using phosphatase activity assays with phosphoprotein substrates, we found that both phosphatases integrate substrate recognition with activating signals to control metal-cofactor binding and substrate dephosphorylation. This integrated control provides a mechanism for PP2C family of phosphatases to produce specific responses by acting on the correct substrates, under the appropriate conditions.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Proteína Fosfatase 2C/metabolismo , Regulação Alostérica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Fosfoproteínas , Fosforilação , Conformação Proteica , Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/genética , Transdução de Sinais , Especificidade por Substrato
16.
Arch Microbiol ; 203(6): 3033-3044, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33782718

RESUMO

The rise of pollution due to the dye industries and textile wastes are evolving rapidly every day. The dyes are used in different trade names by the textile industries. The actual chemistry of dye is vague and difficult to understand even today though we are equipped technically. The toxic effects of the dyes and the reasons behind the acute toxicity are also an undiscovered mystery; therefore, no effective measures can be employed to degrade dyes. Deploying physical or chemical methods to pre-treat the azo dyes are expensive, extremely energy-consuming, and are not environment friendly. Hence, the use of microbes for textile dye degradation will be eco-friendly and is probably a cost-effective alternative to physicochemical methods. The present study was conducted to investigate the degradation of azo dyes isolated from textile effluent contaminated soil by employing the bacterial strains for degradation. The bacterial strains could degrade the optimum concentration of mixed azo dyes (200 mg/L) with an incubation up to 5 days. The decolourization of the dyes was expressed in terms of percentage of decolourization, and was found that about 87.35% of degradation by Bacillus subtilis strain. The enzyme responsible was analyzed as intracellular azoreductase involved in the degradation of mixed azo dyes. The enzymatic pathway and 1-phenyl-2-4(4-methyl phenyl)-diazene 1-oxide was observed as the major metabolite by GC-MS analysis. The in silico study determined the binding of mixed azo dye with azoreductase and hypothesized that their linking could be the main reason for the degradation of mixed azo dye.


Assuntos
Compostos Azo , Bacillus subtilis , Biodegradação Ambiental , Nitrorredutases , Compostos Azo/metabolismo , Bacillus subtilis/enzimologia , Simulação de Acoplamento Molecular , Nitrorredutases/metabolismo
17.
Int J Biol Macromol ; 180: 222-233, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33737179

RESUMO

Catalases catalyze the decomposition of hydrogen peroxide into water and oxygen. Limited reports are available on characterization of manganese-catalases. We describe here molecular cloning and expression in Escherichia coli of a putative manganese-catalase gene from mesophilic bacterium, Bacillus subtilis R5. The gene product, CatBsu, produced as a soluble protein, was purified to apparent homogeneity and biochemically characterized. The absorption spectra and nonsignificant inhibition by sodium azide indicated that it is a manganese-catalase. The protein was in homohexameric form in solution, with a subunit molecular weight of 30 kDa, containing ~2 Mn2+ and ~1 Ca2+ per subunit. CatBsu showed highest activity at pH 8.0 and 55 °C. It was found to be highly active with a specific activity of 25,290 µmol min-1 mg-1 and apparent Km and kcat values of 98 mM and 1.27 × 104 s-1 subunit-1, respectively. Although from a mesophilic source, it exhibited a half-life of 2 h at 80 °C. Furthermore, the active site and metal binding residues in CatBsu were predicted by homology modelling and molecular docking. To the best of our knowledge, this is the first characterization of a manganese-catalase from genus Bacillus.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catalase/química , Catalase/metabolismo , Cálcio/metabolismo , Catalase/genética , Domínio Catalítico , Clonagem Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Meia-Vida , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Manganês/metabolismo , Simulação de Acoplamento Molecular , Peso Molecular , Filogenia , Ligação Proteica , Solubilidade
18.
Appl Biochem Biotechnol ; 193(6): 1853-1872, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33644831

RESUMO

α-amylase is known to have antibiofilm activity against biofilms of both Gram positive and Gram-negative bacterial strains. Partially purified α-amylase from Bacillus subtilis was found to have inhibit biofilm formed by P. aeruginosa and S. aureus. The spectrophotometric and microscopic studies revealed that the antibiofilm efficacy of the working strain is greater than commercially purchased α-amylase. Response surface methodology (RSM) and artificial neural network (ANN) help to predict the optimum conditions [pH 8, treatment time 6 h and enzyme concentration (200 µg/mL)] for maximum biofilm eradication. This was confirmed by several in vitro experiments. Molecular docking interactions of α-amylase with the extracellular polymeric substances (EPS) of both P. aeruginosa and S. aureus indicate towards the existence of an efficient energy driven spontaneous process. Thus, this study highlights a combination of experimental and computational approach showing the naturally extracted α-amylase from B. subtilis having the potency of removing the biofilms of harmful bacterial strains involved in causing various nosocomial infections.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias , Biofilmes/efeitos dos fármacos , Redes Neurais de Computação , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/fisiologia , alfa-Amilases , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Biofilmes/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , alfa-Amilases/química , alfa-Amilases/farmacologia
19.
Angew Chem Int Ed Engl ; 60(20): 11448-11456, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33687787

RESUMO

Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and half-life up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50-100 °C. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs- and/or thermal-resistant lipases and other α/ß-hydrolases.


Assuntos
Bacillus subtilis/enzimologia , Lipase/química , Compostos Orgânicos/química , Biocatálise , Estabilidade Enzimática , Lipase/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Sais/química , Solventes/química , Temperatura
20.
Nucleic Acids Res ; 49(8): 4643-4654, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33788929

RESUMO

RNase Y and RNase E are disparate endoribonucleases that govern global mRNA turnover/processing in the two evolutionary distant bacteria Bacillus subtilis and Escherichia coli, respectively. The two enzymes share a similar in vitro cleavage specificity and subcellular localization. To evaluate the potential equivalence in biological function between the two enzymes in vivo we analyzed whether and to what extent RNase E is able to replace RNase Y in B. subtilis. Full-length RNase E almost completely restores wild type growth of the rny mutant. This is matched by a surprising reversal of transcript profiles both of individual genes and on a genome-wide scale. The single most important parameter to efficient complementation is the requirement for RNase E to localize to the inner membrane while truncation of the C-terminal sequences corresponding to the degradosome scaffold has only a minor effect. We also compared the in vitro cleavage activity for the major decay initiating ribonucleases Y, E and J and show that no conclusions can be drawn with respect to their activity in vivo. Our data confirm the notion that RNase Y and RNase E have evolved through convergent evolution towards a low specificity endonuclease activity universally important in bacteria.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Regulação para Baixo , Endorribonucleases/genética , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Evolução Molecular , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas In Vitro , Microscopia de Fluorescência , Ribonucleases/genética , Ribonucleases/metabolismo , Regulação para Cima
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