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1.
Molecules ; 26(13)2021 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-34206529

RESUMEN

Urease is an enzyme that plays a significant role in the hydrolysis of urea into carbonic acid and ammonia via the carbamic acid formation. The resultant increase in pH leads to the onset of various pathologies such as gastric cancer, urolithiasis, hepatic coma, hepatic encephalopathy, duodenal ulcers and peptic ulcers. Urease inhibitors can reduce the urea hydrolysis rate and development of various diseases. The Cinnamomum genus is used in a large number of traditional medicines. It is well established that stem bark of Cinnamomum cassia exhibits antiulcerogenic potential. The present study evaluated the inhibitory effect of seven extracts of Cinnamomum camphora, Cinnamomum verum and two pure compounds Camphene and Cuminaldehyde on urease enzyme. Kinetic studies of potential inhibitors were carried out. Methanol extract (IC50 980 µg/mL) of C. camphora and a monoterpene Camphene (IC50 0.147 µg/mL) possess significant inhibitory activity. The Lineweaver Burk plot analysis suggested the competitive inhibition by methanol extract, hexane fraction and Camphene. The Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of hexane fraction revealed the contribution of various terpenes. The present study targets terpenes as a new class of inhibitors that have potential therapeutic value for further development as novel drugs.


Asunto(s)
Proteínas Bacterianas , Cinnamomum/química , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Extractos Vegetales/química , Ureasa , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Ureasa/antagonistas & inhibidores , Ureasa/química
2.
Molecules ; 26(13)2021 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-34202153

RESUMEN

In this work, we have investigated the binding conformations of the substrate in the active site of 5-HIU hydrolase kpHIUH and its catalytic hydrolysis mechanism. Docking calculations revealed that the substrate adopts a conformation in the active site with its molecular plane laying parallel to the binding interface of the protein dimer of kpHIUH, in which His7 and His92 are located adjacent to the hydrolysis site C6 and have hydrogen bond interactions with the lytic water. Based on this binding conformation, density functional theory calculations indicated that the optimal catalytic mechanism consists of two stages: (1) the lytic water molecule is deprotonated by His92 and carries out nucleophilic attack on C6=O of 5-HIU, resulting in an oxyanion intermediate; (2) by accepting a proton transferred from His92, C6-N5 bond is cleaved to completes the catalytic cycle. The roles of His7, His92, Ser108 and Arg49 in the catalytic reaction were revealed and discussed in detail.


Asunto(s)
Proteínas Bacterianas/química , Hidrolasas/química , Klebsiella pneumoniae/enzimología , Modelos Moleculares , Catálisis , Dominio Catalítico , Ácido Úrico/análogos & derivados , Ácido Úrico/química
3.
Molecules ; 26(13)2021 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-34203222

RESUMEN

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Fluorescentes Verdes/química , Cuerpos de Inclusión/química , Fosfolipasas A1/química , Proteínas Recombinantes de Fusión/química , Yersinia pseudotuberculosis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Fosfolipasas A1/biosíntesis , Fosfolipasas A1/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Yersinia pseudotuberculosis/enzimología
4.
Molecules ; 26(11)2021 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-34204994

RESUMEN

Chlorophylls and bacteriochlorophylls, together with carotenoids, serve, noncovalently bound to specific apoproteins, as principal light-harvesting and energy-transforming pigments in photosynthetic organisms. In recent years, enormous progress has been achieved in the elucidation of structures and functions of light-harvesting (antenna) complexes, photosynthetic reaction centers and even entire photosystems. It is becoming increasingly clear that light-harvesting complexes not only serve to enlarge the absorption cross sections of the respective reaction centers but are vitally important in short- and long-term adaptation of the photosynthetic apparatus and regulation of the energy-transforming processes in response to external and internal conditions. Thus, the wide variety of structural diversity in photosynthetic antenna "designs" becomes conceivable. It is, however, common for LHCs to form trimeric (or multiples thereof) structures. We propose a simple, tentative explanation of the trimer issue, based on the 2D world created by photosynthetic membrane systems.


Asunto(s)
Cianobacterias/metabolismo , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Plantas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transferencia de Energía , Modelos Moleculares , Fotosíntesis , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformación Proteica , Multimerización de Proteína
6.
Nat Commun ; 12(1): 3214, 2021 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-34088904

RESUMEN

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.


Asunto(s)
Proteínas Arqueales/metabolismo , División Celular/fisiología , Methanobrevibacter/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ciclo Celular , División Celular/genética , Secuencia Conservada , Cristalografía por Rayos X , Evolución Molecular , Methanobrevibacter/genética , Methanobrevibacter/ultraestructura , Microscopía Electrónica de Transmisión , Modelos Moleculares , Filogenia , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura
7.
Int J Mol Sci ; 22(11)2021 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-34070642

RESUMEN

Urate oxidase initiates the uric acid degradation pathways and is extensively used for protein drug development for gout therapy and serum uric acid diagnosis. We first present the biochemical and structural elucidation of a urate oxidase from the extremophile microorganism Deinococcus radiodurans (DrUox). From enzyme characterization, DrUox showed optimal catalytic ability at 30 °C and pH 9.0 with high stability under physiological conditions. Only the Mg2+ ion moderately elevated its activity, which indicates the characteristic of the cofactor-free urate oxidase family. Of note, DrUox is thermostable in mesophilic conditions. It retains almost 100% activity when incubated at 25 °C and 37 °C for 24 h. In this study, we characterized a thermostable urate oxidase, DrUox with high catalytic efficiency and thermal stability, which strengthens its potential for medical applications.


Asunto(s)
Proteínas Bacterianas , Deinococcus , Gota/tratamiento farmacológico , Hiperuricemia/tratamiento farmacológico , Urato Oxidasa , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/uso terapéutico , Deinococcus/enzimología , Deinococcus/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Urato Oxidasa/química , Urato Oxidasa/genética , Urato Oxidasa/uso terapéutico
8.
Int J Mol Sci ; 22(9)2021 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-34063039

RESUMEN

KfrC proteins are encoded by the conjugative broad-host-range plasmids that also encode alpha-helical filament-forming KfrA proteins as exemplified by the RA3 plasmid from the IncU incompatibility group. The RA3 variants impaired in kfrA, kfrC, or both affected the host's growth and demonstrated the altered stability in a species-specific manner. In a search for partners of the alpha-helical KfrC protein, the host's membrane proteins and four RA3-encoded proteins were found, including the filamentous KfrA protein, segrosome protein KorB, and the T4SS proteins, the coupling protein VirD4 and ATPase VirB4. The C-terminal, 112-residue dimerization domain of KfrC was involved in the interactions with KorB, the master player of the active partition, and VirD4, a key component of the conjugative transfer process. In Pseudomonas putida, but not in Escherichia coli, the lack of KfrC decreased the stability but improved the transfer ability. We showed that KfrC and KfrA were involved in the plasmid maintenance and conjugative transfer and that KfrC may play a species-dependent role of a switch between vertical and horizontal modes of RA3 spreading.


Asunto(s)
Proteínas Bacterianas/química , Especificidad del Huésped , Plásmidos/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Conjugación Genética , Escherichia coli/genética , Modelos Biológicos , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos , Eliminación de Secuencia
9.
Science ; 372(6547): 1220-1224, 2021 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-34112695

RESUMEN

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme that lacks affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding messenger RNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid that is impermeable to nucleases, and emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing nonviral carriers for diverse vaccine and delivery applications.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cápside/metabolismo , Evolución Molecular Dirigida , ARN Mensajero/metabolismo , Sustitución de Aminoácidos , Aquifex/enzimología , Proteínas Bacterianas/química , Cápside/química , Microscopía por Crioelectrón , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Nucleocápside/química , Nucleocápside/genética , Nucleocápside/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Subunidades de Proteína , ARN Mensajero/química , ARN Mensajero/genética , Ribonucleasas/metabolismo
10.
ACS Appl Mater Interfaces ; 13(23): 26694-26703, 2021 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-34081428

RESUMEN

A long-standing goal in the field of biotechnology is to develop and understand design rules for the stabilization of enzymes upon immobilization to materials. While immobilization has sometimes been successful as a strategy to stabilize enzymes, the design of synthetic materials that stabilize enzymes remains largely empirical. We sought to overcome this challenge by investigating the mechanistic basis for the stabilization of immobilized lipases on random copolymer brush surfaces comprised of poly(ethylene glycol) methacrylate (PEGMA) and sulfobetaine methacrylate (SBMA), which represent novel heterogeneous supports for immobilized enzymes. Using several related but structurally diverse lipases, including Bacillus subtilis lipase A (LipA), Rhizomucor miehei lipase, Candida rugosa lipase, and Candida antarctica lipase B (CALB), we showed that the stability of each lipase at elevated temperatures was strongly dependent on the fraction of PEGMA in the brush layer. This dependence was explained by developing and applying a new algorithm to quantify protein surface hydrophobicity, which involved using unsupervised cluster analysis to identify clusters of hydrophobic atoms. Characterization of the lipases showed that the optimal brush composition correlated with the free energy of solvation per enzyme surface area, which ranged from -17.1 kJ/mol·nm2 for LipA to -11.8 kJ/mol·nm2 for CALB. Additionally, using this algorithm, we found that hydrophobic patches consisting of aliphatic residues had a higher free energy than patches consisting of aromatic residues. By providing the basis for rationally tuning the interface between enzymes and materials, this understanding will transform the use of materials to reliably ruggedize enzymes under extreme conditions.


Asunto(s)
Biotecnología/normas , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Polímeros/química , Polímeros/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biocatálisis , Estabilidad de Enzimas , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas
11.
Phys Rev Lett ; 126(21): 218102, 2021 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-34114848

RESUMEN

We introduce a statistical and linear response theory of selective conduction in biological ion channels with multiple binding sites and possible point mutation. We derive an effective grand-canonical ensemble and generalized Einstein relations for the selectivity filter, assuming strongly coordinated ionic motion, and allowing for ionic Coulomb blockade. The theory agrees well with data from the KcsA K^{+} channel and a mutant. We show that the Eisenman relations for thermodynamic selectivity follow from the condition for fast conduction and find that maximum conduction requires the binding sites to be nearly identical.


Asunto(s)
Canales Iónicos/química , Canales Iónicos/genética , Modelos Biológicos , Modelos Químicos , Mutación Puntual , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Fenómenos Biofísicos , Canales Iónicos/metabolismo , Modelos Genéticos , Modelos Moleculares , Canales de Potasio/química , Canales de Potasio/genética , Canales de Potasio/metabolismo , Termodinámica
12.
Sci Rep ; 11(1): 12410, 2021 06 14.
Artículo en Inglés | MEDLINE | ID: mdl-34127732

RESUMEN

In situ generation of antibacterial and antiviral agents by harnessing the catalytic activity of enzymes on surfaces provides an effective eco-friendly approach for disinfection. The perhydrolase (AcT) from Mycobacterium smegmatis catalyzes the perhydrolysis of acetate esters to generate the potent disinfectant, peracetic acid (PAA). In the presence of AcT and its two substrates, propylene glycol diacetate and H2O2, sufficient and continuous PAA is generated over an extended time to kill a wide range of bacteria with the enzyme dissolved in aqueous buffer. For extended self-disinfection, however, active and stable AcT bound onto or incorporated into a surface coating is necessary. In the current study, an active, stable and reusable AcT-based coating was developed by incorporating AcT into a polydopamine (PDA) matrix in a single step, thereby forming a biocatalytic composite onto a variety of surfaces. The resulting AcT-PDA composite coatings on glass, metal and epoxy surfaces yielded up to 7-log reduction of Gram-positive and Gram-negative bacteria when in contact with the biocatalytic coating. This composite coating also possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes. The single-step approach enables rapid and facile fabrication of enzyme-based disinfectant composite coatings with high activity and stability, which enables reuse following surface washing. As a result, this enzyme-polymer composite technique may serve as a general strategy for preparing antibacterial and antiviral surfaces for applications in health care and common infrastructure safety, such as in schools, the workplace, transportation, etc.


Asunto(s)
Antibacterianos/química , Antivirales/química , Proteínas Bacterianas/química , Hidrolasas/química , Indoles/química , Polímeros/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antivirales/metabolismo , Antivirales/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , COVID-19/patología , COVID-19/virología , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/metabolismo , Materiales Biocompatibles Revestidos/farmacología , Estabilidad de Medicamentos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Hidrolasas/genética , Hidrolasas/metabolismo , Cinética , Mycobacterium smegmatis/enzimología , Ácido Peracético/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , SARS-CoV-2/efectos de los fármacos
13.
Int J Biol Macromol ; 183: 2354-2363, 2021 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-34081954

RESUMEN

DarR, a novel member of the LTTR family derived from Vibrio fischeri, activates transcription in response to d-Asp and regulates the overexpression of the racD genes encoding a putative aspartate racemase, RacD. Here, the crystal structure of full-length DarR and its mutant DarR-M202I were obtained by X-ray crystallography. According to the electron density map analysis of full-length DarR, the effector binding site of DarR is occupied by 2-Morpholinoethanesulfonic acid monohydrate (MES), which could interact with amino acids in the effector binding site and stabilize the effector binding site. Furthermore, we elaborated the structure of DarR-M202I, where methionine is replaced by isoleucine resulting in overexpression of the downstream operon. By comparing DarR-MES and DarR-M202I, we found similar behavior of DarR-MES in terms of the stability of the RD active pocket and the deflection angle of the DBD. The Isothermal titration calorimetry and Gel-filtration chromatography experiments showed that only when the target DNA sequence of a particular quasi-palindromic sequence exceeds 19 bp, DarR can effectively bind to racD promoter. This study will help enhance our understanding of the mechanism in the transcriptional regulation of LTTR family transcription factors.


Asunto(s)
Aliivibrio fischeri/metabolismo , Proteínas Bacterianas/metabolismo , Factores de Transcripción/metabolismo , Aliivibrio fischeri/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Ácido D-Aspártico/metabolismo , Regulación Bacteriana de la Expresión Génica , Simulación de Dinámica Molecular , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética
14.
Nat Commun ; 12(1): 3577, 2021 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-34117249

RESUMEN

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.


Asunto(s)
Antibacterianos/farmacología , Diterpenos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Lincosamidas/farmacología , Compuestos Policíclicos/farmacología , Estreptograminas/farmacología , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Farmacorresistencia Bacteriana/genética , Bacterias Grampositivas/genética , Modelos Moleculares , Peptidil Transferasas/metabolismo , Conformación Proteica , ARN Mensajero , Ribosomas/metabolismo
15.
Nucleic Acids Res ; 49(11): 6569-6586, 2021 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-34107018

RESUMEN

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.


Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , AdnB Helicasas/química , Vibrio cholerae/enzimología , Proteínas Bacterianas/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , AdnB Helicasas/metabolismo , Modelos Moleculares , Conformación Proteica , Serina/química
16.
Nucleic Acids Res ; 49(11): 6587-6595, 2021 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-34107040

RESUMEN

Bacteriophages have evolved a range of anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes, therefore Acrs can be used as switches to regulate gene editing. Herein, we report the crystal structure of a quaternary complex of AcrIIA14 bound SauCas9-sgRNA-dsDNA at 2.22 Å resolution, revealing the molecular basis for AcrIIA14 recognition and inhibition. Our structural and biochemical data analysis suggest that AcrIIA14 binds to a non-conserved region of SauCas9 HNH domain that is distinctly different from AcrIIC1 and AcrIIC3, with no significant effect on sgRNA or dsDNA binding. Further, our structural data shows that the allostery of the HNH domain close to the substrate DNA is sterically prevented by AcrIIA14 binding. In addition, the binding of AcrIIA14 triggers the conformational allostery of the HNH domain and the L1 linker within the SauCas9, driving them to make new interactions with the target-guide heteroduplex, enhancing the inhibitory ability of AcrIIA14. Our research both expands the current understanding of anti-CRISPRs and provides additional culues for the rational use of the CRISPR-Cas system in genome editing and gene regulation.


Asunto(s)
Proteínas Bacterianas/química , Proteína 9 Asociada a CRISPR/antagonistas & inhibidores , Proteína 9 Asociada a CRISPR/química , Staphylococcus aureus/enzimología , Regulación Alostérica , Cristalografía por Rayos X , ADN/química , Modelos Moleculares , Dominios Proteicos , ARN/química
17.
Int J Mol Sci ; 22(11)2021 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-34072989

RESUMEN

Under anaerobic conditions, bacteria may utilize nitrates and nitrites as electron acceptors. Sensitivity to nitrous compounds is achieved via several mechanisms, some of which rely on sensor histidine kinases (HKs). The best studied nitrate- and nitrite-sensing HKs (NSHKs) are NarQ and NarX from Escherichia coli. Here, we review the function of NSHKs, analyze their natural diversity, and describe the available structural information. In particular, we show that around 6000 different NSHK sequences forming several distinct clusters may now be found in genomic databases, comprising mostly the genes from Beta- and Gammaproteobacteria as well as from Bacteroidetes and Chloroflexi, including those from anaerobic ammonia oxidation (annamox) communities. We show that the architecture of NSHKs is mostly conserved, although proteins from Bacteroidetes lack the HAMP and GAF-like domains yet sometimes have PAS. We reconcile the variation of NSHK sequences with atomistic models and pinpoint the structural elements important for signal transduction from the sensor domain to the catalytic module over the transmembrane and cytoplasmic regions spanning more than 200 Å.


Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas , Histidina Quinasa , Proteínas de la Membrana , Nitratos/metabolismo , Nitritos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Histidina Quinasa/química , Histidina Quinasa/clasificación , Histidina Quinasa/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Dominios Proteicos
18.
Int J Mol Sci ; 22(11)2021 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-34073028

RESUMEN

In the current work we study, via molecular simulations and experiments, the folding and stability of proteins from the tertiary motif of 4-α-helical bundles, a recurrent motif consisting of four amphipathic α-helices packed in a parallel or antiparallel fashion. The focus is on the role of the loop region in the structure and the properties of the wild-type Rop (wtRop) and RM6 proteins, exploring the key factors which can affect them, through all-atom molecular dynamics (MD) simulations and supporting by experimental findings. A detailed investigation of structural and conformational properties of wtRop and its RM6 loopless mutation is presented, which display different physical characteristics even in their native states. Then, the thermal stability of both proteins is explored showing RM6 as more thermostable than wtRop through all studied measures. Deviations from native structures are detected mostly in tails and loop regions and most flexible residues are indicated. Decrease of hydrogen bonds with the increase of temperature is observed, as well as reduction of hydrophobic contacts in both proteins. Experimental data from circular dichroism spectroscopy (CD), are also presented, highlighting the effect of temperature on the structural integrity of wtRop and RM6. The central goal of this study is to explore on the atomic level how a protein mutation can cause major changes in its physical properties, like its structural stability.


Asunto(s)
Proteínas Bacterianas/química , Pliegue de Proteína , Proteínas de Unión al ARN/química , Secuencia de Aminoácidos , Enlace de Hidrógeno , Conformación Proteica en Hélice alfa , Estabilidad Proteica , Estructura Terciaria de Proteína , Temperatura
19.
ACS Chem Biol ; 16(6): 1040-1049, 2021 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-34105348

RESUMEN

O-GlcNAcylation is an O-linked ß-N-acetyl-glucosamine (O-GlcNAc)-monosaccharide modification of serine or threonine in proteins that plays a vital role in many critical cellular processes. Owing to its low molecular weight, uncharged property, and difficulty in distinguishing from ß-N-acetyl-galactosamine (GalNAc), the lack of high specificity and avidity tools and sophisticated quantification methods have always been the bottleneck in analyzing O-GlcNAc functions. Here, we compared glycan array data of the mutant of Clostridium perfringen OGA (CpOGAD298N), O-GlcNAc antibody CTD110.6, and several lectins. We found that CpOGAD298N can effectively distinguish GlcNAc from GalNAc. Glycan array analysis and isothermal titration calorimetry (ITC) show that CpOGAD298N has a GlcNAc specific binding characteristic. CpOGAD298N could be used in far-western, flow cytometry analysis, and confocal imaging to demonstrate the existence of O-GlcNAc proteins. Using the CpOGAD298N affinity column, we identified 84 highly confident O-GlcNAc modified peptides from 82 proteins in the MCF-7 cell line and 33 highly confident peptides in 33 proteins from mouse liver tissue; most of them are novel O-GlcNAc proteins and could not bind with wheat germ agglutinin (WGA). Besides being used as a facile enrichment tool, a combination of CpOGAD298N with the proximity ligation assay (PLA) is successfully used to quantify O-GlcNAc modified histone H2B, which is as low as femtomoles in MCF-7 cell lysate. These results suggest that CpOGAD298N is a specific tool for detection (far-western, flow cytometry analysis, and confocal imaging) and enrichment of O-GlcNAcylated proteins and peptides, and the CpOGAD298N-PLA method is useful for quantifying certain O-GlcNAc protein.


Asunto(s)
Acetilglucosamina/metabolismo , Proteínas Bacterianas/metabolismo , Clostridium perfringens/metabolismo , Acetilglucosamina/análisis , Acilación , Proteínas Bacterianas/química , Clostridium perfringens/química , Glicosilación , Polisacáridos/análisis , Polisacáridos/metabolismo
20.
Nat Commun ; 12(1): 3867, 2021 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-34162839

RESUMEN

Enzymes can evolve new catalytic activity when environmental changes present them with novel substrates. Despite this seemingly straightforward relationship, factors other than the direct catalytic target can also impact adaptation. Here, we characterize the catalytic activity of a recently evolved bacterial methyl-parathion hydrolase for all possible combinations of the five functionally relevant mutations under eight different laboratory conditions (in which an alternative divalent metal is supplemented). The resultant adaptive landscapes across this historical evolutionary transition vary in terms of both the number of "fitness peaks" as well as the genotype(s) at which they are found as a result of genotype-by-environment interactions and environment-dependent epistasis. This suggests that adaptive landscapes may be fluid and molecular adaptation is highly contingent not only on obvious factors (such as catalytic targets), but also on less obvious secondary environmental factors that can direct it towards distinct outcomes.


Asunto(s)
Adaptación Fisiológica/genética , Bacterias/genética , Proteínas Bacterianas/genética , Epistasis Genética , Hidrolasas/genética , Secuencia de Aminoácidos , Bacterias/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biocatálisis , Evolución Molecular , Interacción Gen-Ambiente , Genotipo , Hidrolasas/química , Hidrolasas/metabolismo , Cinética , Metales/química , Metales/metabolismo , Metil Paratión/química , Metil Paratión/metabolismo , Mutación , Dominios Proteicos , Homología de Secuencia de Aminoácido
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