scholarly journals A Novel Mercuric Reductase from the Unique Deep Brine Environment of Atlantis II in the Red Sea

2013 ◽  
Vol 289 (3) ◽  
pp. 1675-1687 ◽  
Author(s):  
Ahmed Sayed ◽  
Mohamed A. Ghazy ◽  
Ari J. S. Ferreira ◽  
João C. Setubal ◽  
Felipe S. Chambergo ◽  
...  
Keyword(s):  
Red Sea ◽  
Three Dimensional ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Mercuric Reductase ◽  
Three Dimensional Modeling ◽  
High Concentrations ◽  
Convective Layer

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg2+, and efficiently detoxifies Hg2+in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.

scholarly journals Structural features of the fly chromatin colors revealed by automatic three-dimensional modeling.

2016 ◽  
Author(s):  
François Serra ◽  
Davide Baù ◽  
Guillaume Filion ◽  
Marc A. Marti-Renom
Keyword(s):  
Spatial Organization ◽  
Three Dimensional ◽  
Structural Features ◽  
Three Dimensions ◽  
Chromosome Conformation ◽  
Three Dimensional Modeling ◽  
Genomic Technologies ◽  
A Genome ◽  
Contact Data ◽  
Three Dimensional Models

The sequence of a genome is insufficient to understand all genomic processes carried out in the cell nucleus. To achieve this, the knowledge of its three- dimensional architecture is necessary. Advances in genomic technologies and the development of new analytical methods, such as Chromosome Conformation Capture (3C) and its derivatives, now permit to investigate the spatial organization of genomes. However, inferring structures from raw contact data is a tedious process for shortage of available tools. Here we present TADbit, a computational framework to analyze and model the chromatin fiber in three dimensions. To illustrate the use of TADbit, we automatically modeled 50 genomic domains from the fly genome revealing differential structural features of the previously defined chromatin colors, establishing a link between the conformation of the genome and the local chromatin composition. More generally, TADbit allows to obtain three-dimensional models ready for visualization from 3C-based experiments and to characterize their relation to gene expression and epigenetic states. TADbit is open-source and available for download from http://www.3DGenomes.org.

An algorithm for estimation of chromosome motion in Four-Dimensional microscopic images

1994 ◽  
Vol 52 ◽  
pp. 930-931
Author(s):  
Wallace Marshall ◽  
David Agard ◽  
John Sedat
Keyword(s):  
Nuclear Organization ◽  
Three Dimensional ◽  
Estimation Algorithm ◽  
Structural Features ◽  
Random Motion ◽  
Structural Representation ◽  
Functional Roles ◽  
The Face ◽  
Chromosome Motion

Analysis of the three-dimensional organization of chromosomes within the nucleus has revealed a number of characteristic structural features. Yet imaging of living nuclei indicate that chromosomes undergo considerable random motion. Maintenance of nuclear organization in the face of such motion is thought to involve the attachment of chromosomes to the nuclear envelope or matrix. Yet while such attachments have been proposed to play a variety of functional roles as well as maintain nuclear organization, direct evidence for the existence of these mechanical interactions in vivo has been lacking. One way to demonstrate such attachment directly would be to estimate the motion of chromosomes and attempt thereby to demonstrate the presence of fixed points, which would indicate attachment of chromatin to some fixed superstructure.We have previously presented a motion estimation algorithm that is designed for tracking the motion of nonrigid and featureless objects such as chromosomes. This algorithm starts with a structural representation for the set of chromosomes at each time point, and then finds a correspondence between elements of the representations at successive time points.

scholarly journals Micro-Structured Patches for Dermal Regeneration Obtained via Electrophoretic Replica Deposition

2020 ◽  
Vol 10 (14) ◽  
pp. 5010
Author(s):  
Arash Ghalayani Esfahani ◽  
Lina Altomare ◽  
Lorenzo Bonetti ◽  
Fereshteh Nejaddehbashi ◽  
Francesca Boccafoschi ◽  
...  
Keyword(s):  
Normal Skin ◽  
Three Dimensional ◽  
Experimental Period ◽  
Structural Features ◽  
Skin Wound ◽  
Hair Follicles ◽  
Artificial Substrates ◽  
Lattice Structures ◽  
Random Porosity

Artificial substrates supporting the healing of skin wounds require specific structural and chemical architectures that promote a recapitulation of the complexity of the native organ. Bottom-up fabrication technologies are emerging as effective strategies to fine tune biochemical, morphological, and structural features intended for regenerative applications. Here, we proposed an electrophoretic replica deposition (EPrD) approach to realize chitosan three-dimensional structures specifically designed to treat patients with serious cutaneous damages or losses. The EPrD process has been optimized to consistently obtain random porosity vs. hierarchical lattice structures, showing mechanical properties in the range of skin tissue (E = 0.2–20 MPa). The obtained patches were tested in vivo via a one-stage grafting procedure in a full thickness skin wound rat model. Chitosan patches showed no adverse reactions throughout the experimental period (14 days). Hair follicles and sebaceous glands were observed in histological sections, indicating the regeneration of a thin epidermal layer with more skin appendages. Immunohistochemistry results demonstrated that keratin 10 was mostly expressed in basal and suprabasal layers, like normal skin, in structures with random porosity and with smaller lattice structures. The obtained results show the potential of EPrD to innovate the design of artificial substrates in skin healing therapies.

DEEP STRUCTURE OF THE FAULT ZONE IN THE MUKHOR-TARKATA SITE OF THE CHUYA DEPRESSION ACCORDING TO NON-STATIONARY ELECTROMAGNETIC SOUNDING DATA USING THREE-DIMENSIONAL MODELING

2021 ◽  
pp. 67-73
Author(s):  
A. M. Sanchaa ◽  
◽  
N. N. Nevedrova ◽  
N. V. Shtabel ◽  
◽  
...  
Keyword(s):  
Fault Zone ◽  
Time Domain ◽  
Deep Structure ◽  
Near Field ◽  
Three Dimensional ◽  
Structural Features ◽  
Electromagnetic Sounding ◽  
Three Dimensional Modeling ◽  
Dimensional Modeling ◽  
Time Domain Electromagnetic

The paper presents the results of three-dimensional modeling of the fault structure in the central part of the Chuya depression in Gornyi Altai within the Mukhor-Tarkhata plot. On this site, from 2004 to the present, researchers of the IPGG SB RAS have been conducting regular annual observations by the method of nearfield time-domain electromagnetic sounding (TSB) to observe the process of restoration of the geological environment after the catastrophic Chuya earthquake with a magnitude of 7.3 in 2003. One of the aftereffects of the destructive earthquake is fracture zones expressed on the surface. Numerous deformations of the surface and industrial objects are observed in the area of the Mukhor-Tarkhata village. The fault zone with sub-vertical fracturing, identified at the site by geological and electromagnetic data, was activated after the earthquake. Detailed data on the geoelectric structure are required for the interpretation and analysis of monitoring data. Based on the interpretation of the near-field time-domain electromagnetic sounding data, the deep structure of the fault zone was obtained. To verify and clarify structural features of the geoelectric model, three-dimensional modeling was performed.

The Potential of Frog Skin Peptides for Anti-Infective Therapies: The Case of Esculentin-1a(1-21)NH2

2020 ◽  
Vol 27 (9) ◽  
pp. 1405-1419 ◽  
Author(s):  
Bruno Casciaro ◽  
Floriana Cappiello ◽  
Maria Rosa Loffredo ◽  
Francesca Ghirga ◽  
Maria Luisa Mangoni
Keyword(s):  
Frog Skin ◽  
Contact Lenses ◽  
Wide Spectrum ◽  
Structural Features ◽  
Biological Properties ◽  
Antibacterial Surface ◽  
High Concentrations

Antimicrobial Peptides (AMPs) are the key effectors of the innate immunity and represent promising molecules for the development of new antibacterial drugs. However, to achieve this goal, some problems need to be overcome: (i) the cytotoxic effects at high concentrations; (ii) the poor biostability and (iii) the difficulty in reaching the target site. Frog skin is one of the richest natural storehouses of AMPs, and over the years, many peptides have been isolated from it, characterized and classified into several families encompassing temporins, brevinins, nigrocins and esculentins. In this review, we summarized how the isolation/characterization of peptides belonging to the esculentin-1 family drove us to the design of an analogue, i.e. esculentin-1a(1-21)NH2, with a powerful antimicrobial action and immunomodulatory properties. The peptide had a wide spectrum of activity, especially against the opportunistic Gram-negative bacterium Pseudomonas aeruginosa. We described the structural features and the in vitro/in vivo biological characterization of this peptide as well as the strategies used to improve its biological properties. Among them: (i) the design of a diastereomer carrying Damino acids in order to reduce the peptide’s cytotoxicity and improve its half-life; (ii) the covalent conjugation of the peptide to gold nanoparticles or its encapsulation into poly(lactide- co-glycolide) nanoparticles; and (iii) the peptide immobilization to biomedical devices (such as silicon hydrogel contact lenses) to obtain an antibacterial surface able to reduce microbial growth and attachment. Summing up the best results obtained so far, this review traces all the steps that led these frog-skin AMPs to the direction of peptide-based drugs for clinical use.

scholarly journals Virtual Screening of Natural Products against Type II Transmembrane Serine Protease (TMPRSS2), the Priming Agent of Coronavirus 2 (SARS-CoV-2)

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2271 ◽  
Author(s):  
Noor Rahman ◽  
Zarrin Basharat ◽  
Muhammad Yousuf ◽  
Giuseppe Castaldo ◽  
Luca Rastrelli ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Three Dimensional ◽  
Natural Compounds ◽  
Structural Features ◽  
Host Cells ◽  
Dimensional Structure ◽  
Active Site Residues ◽  
Transmembrane Serine Protease

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused about 2 million infections and is responsible for more than 100,000 deaths worldwide. To date, there is no specific drug registered to combat the disease it causes, named coronavirus disease 2019 (COVID-19). In the current study, we used an in silico approach to screen natural compounds to find potent inhibitors of the host enzyme transmembrane protease serine 2 (TMPRSS2). This enzyme facilitates viral particle entry into host cells, and its inhibition blocks virus fusion with angiotensin-converting enzyme 2 (ACE2). This, in turn, restricts SARS-CoV-2 pathogenesis. A three-dimensional structure of TMPRSS2 was built using SWISS-MODEL and validated by RAMPAGE. The natural compounds library Natural Product Activity and Species Source (NPASS), containing 30,927 compounds, was screened against the target protein. Two techniques were used in the Molecular Operating Environment (MOE) for this purpose, i.e., a ligand-based pharmacophore approach and a molecular docking-based screening. In total, 2140 compounds with pharmacophoric features were retained using the first approach. Using the second approach, 85 compounds with molecular docking comparable to or greater than that of the standard inhibitor (camostat mesylate) were identified. The top 12 compounds with the most favorable structural features were studied for physicochemical and ADMET (absorption, distribution, metabolism, excretion, toxicity) properties. The low-molecular-weight compound NPC306344 showed significant interaction with the active site residues of TMPRSS2, with a binding energy score of −14.69. Further in vitro and in vivo validation is needed to study and develop an anti-COVID-19 drug based on the structures of the most promising compounds identified in this study.

scholarly journals Site-directed mutagenesis of a Saccharomyces cerevisiae mitochondrial translation initiation codon.

Genetics ◽  
1991 ◽  
Vol 129 (3) ◽  
pp. 659-668 ◽  
Author(s):  
L S Folley ◽  
T D Fox
Keyword(s):  
Translation Initiation ◽  
Structural Features ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Initiation Codon ◽  
Translation Initiation Site ◽  
Wild Type ◽  
Mitochondrial Translation ◽  
Translation Initiation Codon

Abstract We have used a generally applicable strategy for gene replacement in yeast mitochondria to mutate the translation initiation codon of the COX3 gene from AUG to AUA. The mutation, cox3-1, substantially reduced, but did not eliminate, translation of cytochrome c oxidase subunit III (coxIII). Strains bearing the mutation exhibited a leaky (partial) nonrespiratory growth phenotype and a reduced incorporation of radiolabeled amino acids into coxIII in vivo in the presence of cycloheximide. Hybridization experiments demonstrated that the mutation had little or no effect on levels of the COX3 mRNA. Residual translation of the cox3-1 mutant mRNA was dependent upon the three nuclearly coded mRNA-specific activators PET494, PET54 and PET122, known from previous studies to work through a site (or sites) upstream of the initiation codon to promote translation of the wild-type mRNA. Furthermore, respiratory growth of cox3-1 mutant strains was sensitive to decreased dosage of genes PET494 and PET122 in heterozygous mutant diploids, unlike the growth of strains carrying wild-type mtDNA. Some residual translation of the cox3-1 mRNA appeared to initiate at the mutant AUA codon, despite the fact that the 610-base 5'-mRNA leader contains numerous AUA triplets. We conclude that, while AUG is an important component of the COX3 translation initiation site, the site probably is also specified by other sequence or structural features.

scholarly journals Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls

2020 ◽  
Vol 477 (23) ◽  
pp. 4635-4654
Author(s):  
Milan Wiesselmann ◽  
Stefanie Hebecker ◽  
José M. Borrero-de Acuña ◽  
Manfred Nimtz ◽  
David Bollivar ◽  
...  
Keyword(s):  
Rhodobacter Capsulatus ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Homology Model ◽  
Subcellular Fractionation ◽  
Site Directed Mutagenesis ◽  
Bioinformatics Analyses ◽  
Monomethyl Ester

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

scholarly journals Amylolytic enzymes - focus on the alpha-amylases from Archae and plants

2021 ◽  
Vol 9 (1) ◽  
pp. 5-26
Author(s):  
Štefan Janeček
Keyword(s):  
Three Dimensional ◽  
Structural Features ◽  
Catalytic Triad ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolases ◽  
Amylolytic Enzymes ◽  
Conserved Sequence ◽  
Glycosidic Bonds ◽  
Evolutionary Relatedness ◽  
Tim Barrel

Amylolytic enzymes represent a group of starch hydrolases and related enzymes that are active towards the α-glycosidic bonds in starch and related poly- and oligosaccharides. The three best known amylolytic enzymes are α-amylase, β-amylase and glucoamylase that, however, differ from each other by their amino acid sequences, three-dimensional structures, reaction mechanisms and catalytic machineries. In the sequence-based classification of all glycoside hydrolases (GHs) they have therefore been classified into the three independent families: GH13 (α-amylases), GH14 (β-amylases) and GH15 (glucoamylases). Some amylolytic enzymes have been placed to the families GH31 and GH57. The family GH13 together with the families GH70 and GH77 constitutes the clan GH-H, well-known as the α-amylase family. It contains more than 6,000 sequences and covers 30 various enzyme specificities sharing the conserved sequence regions, catalytic TIM-barrel fold, retaining reaction mechanism and catalytic triad. Among the GH13 α-amylases, those produced by plants and archaebacteria exhibit common sequence similarities that distinguish them from the α-amylases of the remaining taxonomic sources. Despite the close evolutionary relatedness between the plant and archaeal α-amylases, there are also specific differences that discriminate them from each other. These specific differences could be used in an effort to reveal the sequence-structural features responsible for the high thermostability of the α-amylases from Archaea.

scholarly journals Thermal Stability of a Mercuric Reductase from the Red Sea Atlantis II Hot Brine Environment as Analyzed by Site-Directed Mutagenesis

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Mohamad Maged ◽  
Ahmed El Hosseiny ◽  
Mona Kamal Saadeldin ◽  
Ramy K. Aziz ◽  
Eman Ramadan
Keyword(s):  
Thermal Stability ◽  
Red Sea ◽  
Structure Prediction ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Parent Molecule ◽  
Mercuric Reductase ◽  
Strong Potential ◽  
Brine Pool

ABSTRACTThe lower convective layer (LCL) of the Atlantis II brine pool of the Red Sea is a unique environment in terms of high salinity, temperature, and high concentrations of heavy metals. Mercuric reductase enzymes functional in such extreme conditions could be considered a potential tool in the environmental detoxification of mercurial poisoning and might alleviate ecological hazards in the mining industry. Here, we constructed a mercuric reductase library from Atlantis II, from which we identified genes encoding two thermostable mercuric reductase (MerA) isoforms: one is halophilic (designated ATII-LCL) while the other is not (designated ATII-LCL-NH). The ATII-LCL MerA has a short motif composed of four aspartic acids (4D414–417) and two characteristic signature boxes that played a crucial role in its thermal stability. To further understand the mechanism behind the thermostability of the two studied enzymes, we mutated the isoform ATII-LCL-NH and found that the substitution of 2 aspartic acids (2D) at positions 415 and 416 enhanced the thermal stability, while other mutations had the opposite effect. The 2D mutant showed superior thermal tolerance, as it retained 81% of its activity after 10 min of incubation at 70°C. A three-dimensional structure prediction revealed newly formed salt bridges and H bonds in the 2D mutant compared to the parent molecule. To the best of our knowledge, this study is the first to rationally design a mercuric reductase with enhanced thermal stability, which we propose to have a strong potential in the bioremediation of mercurial poisoning.IMPORTANCEThe Red Sea is an attractive environment for bioprospecting. There are 25 brine-filled deeps in the Red Sea. The Atlantis II brine pool is the biggest and hottest of such hydrothermal ecosystems. We generated an environmental mercuric reductase library from the lowermost layer of the Atlantis II brine pool, in which we identified two variants of the mercuric reductase enzyme (MerA). One is the previously described halophilic and thermostable ATII-LCL MerA and the other is a nonhalophilic relatively less thermostable enzyme, designated ATII-LCL-NH MerA. We used the ATII-LCL-NH enzyme as a parent molecule to locate the amino acid residues involved in the noticeably higher thermotolerance of the homolog ATII-LCL MerA. Moreover, we designed a novel enzyme with superior thermal stability. This enzyme might have strong potential in the bioremediation of mercuric toxicity.

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