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dc.contributor.authorVogler, M.
dc.contributor.authorDasSarma, P.
dc.contributor.authorDasSarma, S.
dc.date.accessioned2020-05-26T20:42:00Z
dc.date.available2020-05-26T20:42:00Z
dc.date.issued2020
dc.identifier.urihttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85084524893&doi=10.3389%2ffmicb.2020.00742&partnerID=40&md5=25938278f6a6ab8648d1c722d4d4abc2
dc.identifier.urihttp://hdl.handle.net/10713/12839
dc.description.abstractEnvironments previously thought to be uninhabitable offer a tremendous wealth of unexplored microorganisms and enzymes. In this paper, we present the discovery and characterization of a novel γ-carbonic anhydrase (γ-CA) from the polyextreme Red Sea brine pool Discovery Deep (2141 m depth, 44.8°C, 26.2% salt) by single-cell genome sequencing. The extensive analysis of the selected gene helps demonstrate the potential of this culture-independent method. The enzyme was expressed in the bioengineered haloarchaeon Halobacterium sp. NRC-1 and characterized by X-ray crystallography and mutagenesis. The 2.6 Å crystal structure of the protein shows a trimeric arrangement. Within the γ-CA, several possible structural determinants responsible for the enzyme’s salt stability could be highlighted. Moreover, the amino acid composition on the protein surface and the intra- and intermolecular interactions within the protein differ significantly from those of its close homologs. To gain further insights into the catalytic residues of the γ-CA enzyme, we created a library of variants around the active site residues and successfully improved the enzyme activity by 17-fold. As several γ-CAs have been reported without measurable activity, this provides further clues as to critical residues. Our study reveals insights into the halophilic γ-CA activity and its unique adaptations. The study of the polyextremophilic carbonic anhydrase provides a basis for outlining insights into strategies for salt adaptation, yielding enzymes with industrially valuable properties, and the underlying mechanisms of protein evolution. Copyright 2020 The Authors.en_US
dc.description.sponsorshipKing Abdullah University of Science and Technology, KAUST: 80NSSC19K0463en_US
dc.description.urihttps://doi.org/10.3389/fmicb.2020.00742en_US
dc.language.isoen_USen_US
dc.publisherFrontiers Media S.A.en_US
dc.relation.ispartofFrontiers in Microbiology
dc.subjectextremophilesen_US
dc.subjectextremozymeen_US
dc.subjectgamma-carbonic anhydraseen_US
dc.subjecthalophilesen_US
dc.subjectmutagenesisen_US
dc.subjectsalt adaptationen_US
dc.subjectthermophilesen_US
dc.titleCrystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydraseen_US
dc.typeArticleen_US
dc.identifier.doi10.3389/fmicb.2020.00742


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