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.

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 Molecular Adaptations of Bacterial Mercuric Reductase to the Hypersaline Kebrit Deep in the Red Sea

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Eman Ramadan ◽  
Mohamad Maged ◽  
Ahmed El Hosseiny ◽  
Felipe S. Chambergo ◽  
João C. Setubal ◽  
...  
Keyword(s):  
Amino Acid ◽  
Red Sea ◽  
Compatible Solutes ◽  
Three Dimensional ◽  
Environmental Stressors ◽  
The Other ◽  
Dimensional Structure ◽  
Amino Acid Substitutions ◽  
Dependent Manner ◽  
Osmotic Balance

ABSTRACTThe hypersaline Kebrit Deep brine pool in the Red Sea is characterized by high levels of toxic heavy metals. Here, we describe two structurally related mercuric reductases (MerAs) from this site which were expressed inEscherichia coli. Sequence similarities suggest that both genes are derived from proteobacteria, most likely theBetaproteobacteriaorGammaproteobacteria. We show that one of the enzymes (K35NH) is strongly inhibited by NaCl, while the other (K09H) is activated in a NaCl-dependent manner. We infer from this difference that the two forms might support the detoxification of mercury in bacterial microorganisms that employ the compatible solutes and salt-in strategies, respectively. Three-dimensional structure modeling shows that all amino acid substitutions unique to each type are located outside the domain responsible for formation of the active MerA homodimer, and the vast majority of these are found on the surface of the molecule. Moreover, K09H exhibits the predominance of acidic over hydrophobic side chains that is typical of halophilic salt-dependent proteins. These findings enhance our understanding of how selection pressures imposed by two environmental stressors have endowed MerA enzymes with catalytic properties that can potentially function in microorganisms that utilize distinct mechanisms for osmotic balance in hypersaline environments.IMPORTANCEAnalysis of two structurally homologous but catalytically distinct mercuric reductases from the Kebrit Deep brine in the Red Sea sheds light on the adaptations that enable microorganisms to cope simultaneously with extreme salinity and toxic mercury compounds. One is strongly inhibited by high NaCl concentrations, while the other exhibits NaCl-dependent activation. Their different activity profiles imply that they may derive from bacterial microorganisms that utilize compatible solutes and salt-in strategies, respectively, to maintain osmotic balance. Three-dimensional modeling reveals that regions not involved in formation of the active homodimer are conserved between the two. However, in the NaCl-dependent form, distinct amino acid substitutions are found in areas that are critical for stability in high salt. The work provides insights into how two environmental stressors have shaped the structure of orthologous enzymes through selection and adaptation, enabling them to retain their catalytic function in what may be very different cellular contexts.

scholarly journals Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

2021 ◽  
Vol 9 ◽  
Author(s):  
Jia-Ren Cao ◽  
Fang-Fang Fan ◽  
Chang-Jiang Lv ◽  
Hong-Peng Wang ◽  
Ye Li ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Aspergillus Terreus ◽  
Three Dimensional ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Wild Type ◽  
Enzyme Thermal Stability ◽  
Α Helix ◽  
Dynamics Simulations

Transaminases that promote the amination of ketones into amines are an emerging class of biocatalysts for preparing a series of drugs and their intermediates. One of the main limitations of (R)-selective amine transaminase from Aspergillus terreus (At-ATA) is its weak thermostability, with a half-life (t1/2) of only 6.9 min at 40°C. To improve its thermostability, four important residue sites (E133, D224, E253, and E262) located on the surface of At-ATA were identified using the enzyme thermal stability system (ETSS). Subsequently, 13 mutants (E133A, E133H, E133K, E133R, E133Q, D224A, D224H, D224K, D224R, E253A, E253H, E253K, and E262A) were constructed by site-directed mutagenesis according to the principle of turning the residues into opposite charged ones. Among them, three substitutions, E133Q, D224K, and E253A, displayed higher thermal stability than the wild-type enzyme. Molecular dynamics simulations indicated that these three mutations limited the random vibration amplitude in the two α-helix regions of 130–135 and 148–158, thereby increasing the rigidity of the protein. Compared to the wild-type, the best mutant, D224K, showed improved thermostability with a 4.23-fold increase in t1/2 at 40°C, and 6.08°C increase in T5010. Exploring the three-dimensional structure of D224K at the atomic level, three strong hydrogen bonds were added to form a special “claw structure” of the α-helix 8, and the residues located at 151–156 also stabilized the α-helix 9 by interacting with each other alternately.

A Nabatean port on the Red Sea: note on the pottery finds from al-Qusayr (al-Wajh, Saudi Arabia)

2020 ◽  
Author(s):  
Caroline Durand
Keyword(s):  
Saudi Arabia ◽  
19Th Century ◽  
Red Sea ◽  
Point Of View ◽  
The Other ◽  
Trade Routes ◽  
Other Hand ◽  
Roman Egypt ◽  
Pottery Sherds ◽  
First Time

Al-Qusayr is located 40 km south of modern al-Wajh, roughly 7 km from the eastern Red Sea shore. This site is known since the mid-19th century, when the explorer R. Burton described it for the first time, in particular the remains of a monumental building so-called al-Qasr. In March 2016, a new survey of the site was undertaken by the al-‘Ula–al-Wajh Survey Project. This survey focused not only on al-Qasr but also on the surrounding site corresponding to the ancient settlement. A surface collection of pottery sherds revealed a striking combination of Mediterranean and Egyptian imports on one hand, and of Nabataean productions on the other hand. This material is particularly homogeneous on the chronological point of view, suggesting a rather limited occupation period for the site. Attesting contacts between Mediterranean merchants, Roman Egypt and the Nabataean kingdom, these new data allow a complete reassessment of the importance of this locality in the Red Sea trade routes during antiquity.

scholarly journals A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitomi Nakamura ◽  
Moeka Yoshikawa ◽  
Naoko Oda-Ueda ◽  
Tadashi Ueda ◽  
Takatoshi Ohkuri
Keyword(s):  
Thermal Stability ◽  
Pichia Pastoris ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Binding Activity ◽  
Comprehensive Analysis ◽  
The Other ◽  
Constant Domain ◽  
Intermolecular Disulfide Bond ◽  
Sds Page

AbstractGenerally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

On the presence of Anchistioides willeyi (Borradaile, 1899) (Decapoda: Caridea: Anchistioididae) in the Red Sea

Zootaxa ◽  
2009 ◽  
Vol 2243 (1) ◽  
pp. 53-56 ◽  
Author(s):  
IVAN MARIN
Keyword(s):  
Red Sea ◽  
Type Species ◽  
Specific Form ◽  
The Other ◽  
Valid Species ◽  
Caribbean Region ◽  
Western Atlantic ◽  
Abdominal Somite ◽  
Single Genus ◽  
Pacific Species

The palaemonoid family Anchistioididae Borradaile, 1915 includes a single genus Anchistioides Paulson, 1875 with four known valid species: Anchistioides compressus Paulson, 1875 (type species), A. willeyi (Borradaile, 1899), A. australiensis (Balss, 1921) and A. antiguensis (Schmitt, 1924). Borradaile (1915) suggested two more species within the genus Amphipalaemon Nobili, 1901 (a junior synonym of Anchisitioides Paulson), Amphipalaemon gardineri Borradaile, 1915 (= Anchistioides gardineri) and Amphipalaemon cooperi Borradaile, 1915 (= Anchistioides cooperi) which were later synonomyzed with Anchisitioides willeyi by Gordon (1935), who also suggested their conspecificity with Anchistioides australiensis. At the present time, Anchistioides australiensis is a valid species (Bruce, 1971; Chace & Bruce, 1993) based on specific morphological features such as the presence of sharp postorbital tooth, oblique distal lamela of scaphocerite and sharply produced spines on posterodorsal angles of sixth abdominal somite (see Bruce, 1971: fig. 9). The other Indo-Pacific species, Anchistioides compressus and A. willeyi, can be clearly identified by specific form of scaphocerite, the presence of a well marked blunt postorbital tubercle in A. willeyi which is absent in A. compressus (e.g., Bruce, 1971) and the number of ventral rostral teeth (3-4 large ventral rostral teeth present in A. willeyi while up to 8 small ventral rostral teeth in A. compressus (Paulson, 1875; Gordon, 1935)). Anchistioides antiguensis is clearly separated geographically being known only from the tropical Western Atlantic and Caribbean region (Schmitt, 1924; Holthuis, 1951; Wheeler & Brown, 1968; Martinez-Iglesias, 1986; Markham et al, 1990; Ramos-Porto et al, 1998; Cardoso, 2006).

scholarly journals RNA-Puzzles: A CASP-like evaluation of RNA three-dimensional structure prediction

RNA ◽  
2012 ◽  
Vol 18 (4) ◽  
pp. 610-625 ◽  
Author(s):  
J. A. Cruz ◽  
M.-F. Blanchet ◽  
M. Boniecki ◽  
J. M. Bujnicki ◽  
S.-J. Chen ◽  
...  
Keyword(s):  
Structure Prediction ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure
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