Maged M.Hosseiny A.E.Saadeldin M.K.Aziz R.K.Ramadan E.Department of BiologySchool of Sciences and EngineeringThe American University in CairoNew CairoEgypt; Department of Microbiology and ImmunologyFaculty of PharmacyCairo UniversityCairoEgypt; Faculty of PharmacyThe British University in Egypt (BUE)El ShoroukEgypt; Science and Technology Research CenterSchool of Sciences and EngineeringThe American University in CairoNew CairoEgypt; Faculty of BiotechnologyOctober University for Modern Sciences and Arts6th October CityCairoEgypt2020-01-092020-01-092019992240https://doi.org/10.1128/AEM.02387-18PubMedID30446558https://t.ly/52yxRScopusThe 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-LCLNH). 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. � 2019 American Society for Microbiology.EnglishAtlantis IIBioprospectingBrine poolsExtreme environmentsMerAMercuric reductaseProtein engineeringRed SeaSite-directed mutagenesisThermostableAmino acidsBioremediationChemical bondsDetoxificationEnzymesHeavy metalsMutagenesisStabilityAtlantisBioprospectingBrine poolsExtreme environmentMerAMercuric reductaseProtein engineeringRed seaSite directed mutagenesisThermostableThermodynamic stabilitybioremediationbrinedetoxificationenzymeenzyme activitygenetic analysisheavy metaltemperature toleranceIndian OceanRed Sea [Indian Ocean]bacterial proteinmercuric reductasemercuryoxidoreductasesea wateramino acid sequencebacteriumchemistryecosystemenzyme stabilityenzymologygeneticsheatIndian Oceanisolation and purificationkineticsmetabolismmicrobiologyprotein motifsequence alignmentsite directed mutagenesisAmino Acid MotifsAmino Acid SequenceBacteriaBacterial ProteinsEcosystemEnzyme StabilityHot TemperatureIndian OceanKineticsMercuryMutagenesis, Site-DirectedOxidoreductasesSeawaterSequence AlignmentArticlehttps://doi.org/10.1128/AEM.02387-18PubMedID30446558