Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase
JournalFrontiers in Microbiology
PublisherFrontiers Media S.A.
MetadataShow full item record
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.
SponsorsKing Abdullah University of Science and Technology, KAUST: 80NSSC19K0463
Identifier to cite or link to this itemhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85084524893&doi=10.3389%2ffmicb.2020.00742&partnerID=40&md5=25938278f6a6ab8648d1c722d4d4abc2; http://hdl.handle.net/10713/12839