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1.
Nat Commun ; 12(1): 3214, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088904

RESUMO

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZCTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that previously reported in bacteria. Phylogenetic analyses of SepF and FtsZ from bacteria and archaea indicate that the two proteins may date back to the Last Universal Common Ancestor (LUCA), and we speculate that the archaeal mode of SepF/FtsZ interaction might reflect an ancestral feature. Our results provide insights into the mechanisms of archaeal cell division and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.


Assuntos
Proteínas Arqueais/metabolismo , Divisão Celular/fisiologia , Methanobrevibacter/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo Celular , Divisão Celular/genética , Sequência Conservada , Cristalografia por Raios X , Evolução Molecular , Methanobrevibacter/genética , Methanobrevibacter/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Filogenia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
2.
Nat Commun ; 12(1): 2801, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990569

RESUMO

Photochemical reaction centers are the engines that drive photosynthesis. The reaction center from heliobacteria (HbRC) has been proposed to most closely resemble the common ancestor of photosynthetic reaction centers, motivating a detailed understanding of its structure-function relationship. The recent elucidation of the HbRC crystal structure motivates advanced spectroscopic studies of its excitonic structure and charge separation mechanism. We perform multispectral two-dimensional electronic spectroscopy of the HbRC and corresponding numerical simulations, resolving the electronic structure and testing and refining recent excitonic models. Through extensive examination of the kinetic data by lifetime density analysis and global target analysis, we reveal that charge separation proceeds via a single pathway in which the distinct A0 chlorophyll a pigment is the primary electron acceptor. In addition, we find strong delocalization of the charge separation intermediate. Our findings have general implications for the understanding of photosynthetic charge separation mechanisms, and how they might be tuned to achieve different functional goals.


Assuntos
Proteínas de Bactérias/química , Clostridiales/química , Imageamento Hiperespectral/métodos , Complexo de Proteínas do Centro de Reação Fotossintética/química , Bacterioclorofilas/química , Clorofila A/química , Eletroquímica , Modelos Moleculares , Estrutura Quaternária de Proteína
3.
Nat Commun ; 12(1): 2791, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990582

RESUMO

Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin-membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity.


Assuntos
Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Animais , Toxinas de Bacillus thuringiensis/genética , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Variação Genética , Inseticidas/química , Inseticidas/farmacologia , Lipossomos/química , Modelos Moleculares , Controle Biológico de Vetores , Domínios Proteicos , Estrutura Quaternária de Proteína , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Homologia Estrutural de Proteína
4.
Nat Commun ; 12(1): 2927, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006869

RESUMO

As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate aminoacyl phospholipids to the outer leaflets of bacterial membranes. The function of MprFs enables Staphylococcus aureus and other pathogenic bacteria to acquire resistance to daptomycin and cationic antimicrobial peptides. Here we present cryo-electron microscopy structures of MprF homodimer from Rhizobium tropici (RtMprF) at two different states in complex with lysyl-phosphatidylglycerol (LysPG). RtMprF contains a membrane-embedded lipid-flippase domain with two deep cavities opening toward the inner and outer leaflets of the membrane respectively. Intriguingly, a hook-shaped LysPG molecule is trapped inside the inner cavity with its head group bent toward the outer cavity which hosts a second phospholipid-binding site. Moreover, RtMprF exhibits multiple conformational states with the synthase domain adopting distinct positions relative to the flippase domain. Our results provide a detailed framework for understanding the mechanisms of MprF-mediated modification and translocation of phospholipids.


Assuntos
Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilgliceróis/metabolismo , Fosfolipídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Transporte Biológico , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Lisina/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Fosfatidilgliceróis/química , Fosfolipídeos/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , Rhizobium tropici/genética , Rhizobium tropici/metabolismo
5.
Molecules ; 26(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946907

RESUMO

Biofilms are aggregates of microorganisms anchored to a surface and embedded in a self-produced matrix of extracellular polymeric substances and have been associated with 80% of all bacterial infections in humans. Because bacteria in biofilms are less amenable to antibiotic treatment, biofilms have been associated with developing antibiotic resistance, a problem that urges developing new therapeutic options and approaches. Interfering with quorum-sensing (QS), an important process of cell-to-cell communication by bacteria in biofilms is a promising strategy to inhibit biofilm formation and development. Here we describe and apply an in silico computational protocol for identifying novel potential inhibitors of quorum-sensing, using CviR-the quorum-sensing receptor from Chromobacterium violaceum-as a model target. This in silico approach combines protein-ligand docking (with 7 different docking programs/scoring functions), receptor-based virtual screening, molecular dynamic simulations, and free energy calculations. Particular emphasis was dedicated to optimizing the discrimination ability between active/inactive molecules in virtual screening tests using a target-specific training set. Overall, the optimized protocol was used to evaluate 66,461 molecules, including those on the ZINC/FDA-Approved database and to the Mu.Ta.Lig Virtual Chemotheca. Multiple promising compounds were identified, yielding good prospects for future experimental validation and for drug repurposing towards QS inhibition.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Descoberta de Drogas , Modelos Moleculares , Percepção de Quorum/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Sítios de Ligação , Descoberta de Drogas/métodos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade
6.
Nat Commun ; 12(1): 2702, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976201

RESUMO

Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Iniciação da Transcrição Genética , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , 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 , Elongação da Transcrição Genética
7.
J Med Chem ; 64(9): 5710-5729, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33891818

RESUMO

Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 µM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.


Assuntos
Antibacterianos/química , Proteínas de Bactérias/metabolismo , Pentosiltransferases/metabolismo , Sequência de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/microbiologia , Gastroenteropatias/patologia , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/patologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/enzimologia , Humanos , Hipoxantina Fosforribosiltransferase/química , Hipoxantina Fosforribosiltransferase/metabolismo , Hipoxantinas/química , Hipoxantinas/metabolismo , Hipoxantinas/farmacologia , Hipoxantinas/uso terapêutico , Cinética , Simulação de Dinâmica Molecular , Organofosfonatos/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Pentosiltransferases/química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Alinhamento de Sequência , Relação Estrutura-Atividade
8.
Methods Mol Biol ; 2281: 67-80, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847952

RESUMO

Single-stranded DNA (ssDNA)-binding protein (SSB) is essential for DNA metabolic processes. SSB also binds to many DNA-binding proteins that constitute the SSB interactome. The mechanism through which PriA helicase, an initiator protein in the DNA replication restart process, is stimulated by SSB in Escherichia coli (EcSSB) has been established. However, some Gram-positive bacterial SSBs such as Bacillus subtilis SsbA (a counterpart of EcSSB), Staphylococcus aureus SsbA, SsbB, and SsbC do not activate PriA helicase. Here, we describe some of the methods used in our laboratory to compare SSB-PriA functional and physical interactions in Gram-positive and -negative bacteria.


Assuntos
Bacillus subtilis/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Helicases/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Ligação Proteica , Análise de Sequência de Proteína , Especificidade da Espécie , Ressonância de Plasmônio de Superfície
9.
Food Microbiol ; 98: 103799, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33875225

RESUMO

Vinegar is elaborated using a semi-continuous submerged culture of a complex microbiota of acetic acid bacteria. The genus Komagataeibacter provides much of the proteins of the metaproteome, being K. europaeus the main species working in this environment. In this work, the protein profile of the vinegar microbiota, obtained by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in samples from different cycle times of an acetification process using an alcohol medium, has been used to describe the functional metaproteome throughout the process. The analysis was focused on Komagataeibacter species which supplied about 90% of the metaproteome and particularly K. europaeus which accounts for more than 70%. According to these results, the natural behaviour of a microbial community in vinegar has been predicted at a quantitative proteomic level. The results revealed that most of the identified proteins involved in the metabolism of amino acids, biosynthesis of proteins, and energy production related-metabolic pathways increased their expression throughout the cycle loading phase and afterwards experimented a decrease coming into play other proteins acting against acetic acid stress. These findings may facilitate a better understanding of the microbiota's role and contributing to obtain a quality product.


Assuntos
Ácido Acético/metabolismo , Acetobacteraceae/metabolismo , Proteínas de Bactérias/metabolismo , Microbiota , Acetobacteraceae/química , Acetobacteraceae/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cromatografia Líquida , Etanol/metabolismo , Fermentação , Proteômica , Espectrometria de Massas em Tandem
10.
Biochemistry (Mosc) ; 86(Suppl 1): S24-S37, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33827398

RESUMO

The review focuses on bacterial metallo-ß-lactamases (MßLs) responsible for the inactivation of ß-lactams and associated antibiotic resistance. The diversity of the active site structure in the members of different MßL subclasses explains different mechanisms of antibiotic hydrolysis and should be taken into account when searching for potential MßL inhibitors. The review describes the features of the antibiotic inactivation mechanisms by various MßLs studied by X-ray crystallography, NMR, kinetic measurements, and molecular modeling. The mechanisms of enzyme inhibition for each MßL subclass are discussed.


Assuntos
Domínio Catalítico , Resistência Microbiana a Medicamentos , beta-Lactamases/metabolismo , Antibacterianos/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrólise , Cinética , Modelos Moleculares , Conformação Proteica , beta-Lactamases/química
11.
Biomed Res Int ; 2021: 6616059, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33860041

RESUMO

Background: Helicobacter pylori pathogenicity and disease severity are determined by the tyrosine phosphorylation motifs of CagA protein. This study is aimed at detecting the presence of H. pylori and identifying the CagA tyrosine phosphorylation motifs in Ghanaian patients. Material and Methods. A total of 94 archival genomic DNA samples from gastric biopsies were used for the study, and H. pylori was detected by amplifying the 16S rRNA gene. The 3'-end variable region of the cagA gene was amplified, and the entire 3'-end was sequenced and translated into amino acids. Results: H. pylori was detected in 53.2% (50/94) of the samples, and all the detected bacteria harboured the cagA gene. Two variants of the bacteria were identified based on the size of the amplified cagA gene: 207 bp and 285 bp. The 207 bp and 285 bp variants accounted for 74% and 22%, respectively, and 4% showed both fragments. Translated amino acid sequence of the cagA gene showed EPIYA-A, EPIYA-B, and EPIYA-C (ABC type) motifs, indicating the Western variant. The CagA protein C-terminal showed insertion of amino acids in the sequence flanking the EPIYA-A motif at the N-terminal and a complete deletion of the EPIYA-CC and EPIYA-CCC motifs together with the flanking sequences. Conclusions: H. pylori identified were Western variant (ABC type) with unique amino acid insertions, suggesting unique variants in Ghanaian patients. Further investigation is however required to understand the role of the molecular diversity of the variant in gastric disease outcome.


Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Helicobacter pylori/fisiologia , Estômago/microbiologia , Estômago/patologia , Tirosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Biópsia , Gana , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Fosforilação , RNA Ribossômico 16S/genética , Relação Estrutura-Atividade
12.
Molecules ; 26(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33922133

RESUMO

The increased interest in sequencing cyanobacterial genomes has allowed the identification of new homologs to both the N-terminal domain (NTD) and C-terminal domain (CTD) of the Orange Carotenoid Protein (OCP). The N-terminal domain homologs are known as Helical Carotenoid Proteins (HCPs). Although some of these paralogs have been reported to act as singlet oxygen quenchers, their distinct functional roles remain unclear. One of these paralogs (HCP2) exclusively binds canthaxanthin (CAN) and its crystal structure has been recently characterized. Its absorption spectrum is significantly red-shifted, in comparison to the protein in solution, due to a dimerization where the two carotenoids are closely placed, favoring an electronic coupling interaction. Both the crystal and solution spectra are red-shifted by more than 50 nm when compared to canthaxanthin in solution. Using molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) studies of HCP2, we aim to simulate these shifts as well as obtain insight into the environmental and coupling effects of carotenoid-protein interactions.


Assuntos
Proteínas de Bactérias/química , Cantaxantina/química , Cianobactérias , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos , Teoria Quântica
13.
Nucleic Acids Res ; 49(8): 4386-4401, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33823541

RESUMO

Bacteria persist under constant threat of predation by bacterial viruses (phages). Bacteria-phage conflicts result in evolutionary arms races often driven by mobile genetic elements (MGEs). One such MGE, a phage satellite in Vibrio cholerae called PLE, provides specific and robust defense against a pervasive lytic phage, ICP1. The interplay between PLE and ICP1 has revealed strategies for molecular parasitism allowing PLE to hijack ICP1 processes in order to mobilize. Here, we describe the mechanism of PLE-mediated transcriptional manipulation of ICP1 structural gene transcription. PLE encodes a novel DNA binding protein, CapR, that represses ICP1's capsid morphogenesis operon. Although CapR is sufficient for the degree of capsid repression achieved by PLE, its activity does not hinder the ICP1 lifecycle. We explore the consequences of repression of this operon, demonstrating that more stringent repression achieved through CRISPRi restricts both ICP1 and PLE. We also discover that PLE transduces in modified ICP1-like particles. Examination of CapR homologs led to the identification of a suite of ICP1-encoded homing endonucleases, providing a putative origin for the satellite-encoded repressor. This work unveils a facet of the delicate balance of satellite-mediated inhibition aimed at blocking phage production while successfully mobilizing in a phage-derived particle.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/crescimento & desenvolvimento , DNA Satélite/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Regulação Viral da Expressão Gênica , Sequências Repetitivas Dispersas , Vibrio cholerae/virologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriófagos/genética , Sítios de Ligação , Sistemas CRISPR-Cas , Proteínas do Capsídeo/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/química , Endonucleases/genética , Óperon/genética , Domínios Proteicos , Transdução Genética , Vibrio cholerae/enzimologia , Vibrio cholerae/genética , Vírion/genética , Vírion/crescimento & desenvolvimento
14.
Molecules ; 26(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806095

RESUMO

Over the past several decades, we have observed a very rapid development in the biotechnological use of lactic acid bacteria (LAB) in various branches of the food industry. All such areas of activity of these bacteria are very important and promise enormous economic and industrial successes. LAB are a numerous group of microorganisms that have the ability to ferment sugars into lactic acid and to produce proteolytic enzymes. LAB proteolytic enzymes play an important role in supplying cells with the nitrogen compounds necessary for their growth. Their nutritional requirements in this regard are very high. Lactic acid bacteria require many free amino acids to grow. The available amount of such compounds in the natural environment is usually small, hence the main function of these enzymes is the hydrolysis of proteins to components absorbed by bacterial cells. Enzymes are synthesized inside bacterial cells and are mostly secreted outside the cell. This type of proteinase remains linked to the cell wall structure by covalent bonds. Thanks to advances in enzymology, it is possible to obtain and design new enzymes and their preparations that can be widely used in various biotechnological processes. This article characterizes the proteolytic activity, describes LAB nitrogen metabolism and details the characteristics of the peptide transport system. Potential applications of proteolytic enzymes in many industries are also presented, including the food industry.


Assuntos
Proteínas de Bactérias/química , Lactobacillales/enzimologia , Peptídeo Hidrolases/química
15.
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
16.
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
17.
Nat Commun ; 12(1): 2333, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879791

RESUMO

Acaryochloris marina is one of the cyanobacterial species that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of A. marina photosystem I (PSI) reaction center, determined by cryo-electron microscopy at 2.58 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments distinct from other type I reaction centers. The paired chlorophyll, or special pair (also referred to as P740 in this case), is a dimer of chlorophyll d and its epimer chlorophyll d'. The primary electron acceptor is pheophytin a, a metal-less chlorin. We show the architecture of this PSI reaction center is composed of 11 subunits and we identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/química , Complexo de Proteína do Fotossistema I/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clorofila/química , Clorofila/metabolismo , Microscopia Crioeletrônica , Cianobactérias/genética , Cianobactérias/metabolismo , Transporte de Elétrons , Luz , Modelos Moleculares , Oxigênio/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/metabolismo , Estrutura Quaternária de Proteína , Subunidades Proteicas , Eletricidade Estática
18.
Nat Commun ; 12(1): 2448, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907196

RESUMO

Despite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Divisão Celular , Parede Celular/ultraestrutura , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Expressão Gênica , Hidrólise , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Transporte Proteico
19.
Int J Mol Sci ; 22(7)2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917581

RESUMO

Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore's inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine activation of GerA requires all three subunits, but which binds L-alanine is unknown. To date, how GRs trigger germination is unknown, in particular due to lack of detailed structural information about B subunits. Using homology modelling with molecular dynamics (MD) simulations, we present structural predictions for the integral membrane protein GerAB. These predictions indicate that GerAB is an α-helical transmembrane protein containing a water channel. The MD simulations with free L-alanine show that alanine binds transiently to specific sites on GerAB. These results provide a starting point for unraveling the mechanism of L-alanine mediated signaling by GerAB, which may facilitate early events in spore germination.


Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Membrana Celular/química , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Esporos Bacterianos/química , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Domínios Proteicos , Esporos Bacterianos/metabolismo
20.
Molecules ; 26(7)2021 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-33918211

RESUMO

The role and existence of low-barrier hydrogen bonds (LBHBs) in enzymatic and protein activity has been largely debated. An interesting case is that of the photoactive yellow protein (PYP). In this protein, two short HBs adjacent to the chromophore, p-coumaric acid (pCA), have been identified by X-ray and neutron diffraction experiments. However, there is a lack of agreement on the chemical nature of these H-bond interactions. Additionally, no consensus has been reached on the presence of LBHBs in the active site of the protein, despite various experimental and theoretical studies having been carried out to investigate this issue. In this work, we perform a computational study that combines classical and density functional theory (DFT)-based quantum mechanical/molecular mechanical (QM/MM) simulations to shed light onto this controversy. Furthermore, we aim to deepen our understanding of the chemical nature and dynamics of the protons involved in the two short hydrogen bonds that, in the dark state of PYP, connect pCA with the two binding pocket residues (E46 and Y42). Our results support the existence of a strong LBHB between pCA and E46, with the H fully delocalized and shared between both the carboxylic oxygen of E46 and the phenolic oxygen of pCA. Additionally, our findings suggest that the pCA interaction with Y42 can be suitably described as a typical short ionic H-bond of moderate strength that is fully localized on the phenolic oxygen of Y42.


Assuntos
Proteínas de Bactérias/química , Domínio Catalítico , Fotorreceptores Microbianos/química , Prótons , Elétrons , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Termodinâmica
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