Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase
Environments 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...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-04-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2020.00742/full |
| _version_ | 1819074403614326784 |
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| author | Malvina Vogler Malvina Vogler Ram Karan Dominik Renn Alexandra Vancea Marie-Theres Vielberg Stefan W. Grötzinger Priya DasSarma Shiladitya DasSarma Jörg Eppinger Michael Groll Magnus Rueping |
| author_facet | Malvina Vogler Malvina Vogler Ram Karan Dominik Renn Alexandra Vancea Marie-Theres Vielberg Stefan W. Grötzinger Priya DasSarma Shiladitya DasSarma Jörg Eppinger Michael Groll Magnus Rueping |
| author_sort | Malvina Vogler |
| collection | DOAJ |
| description | Environments 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. |
| first_indexed | 2024-12-21T18:08:58Z |
| format | Article |
| id | doaj.art-e2f6f414774945e7a023b8eb9c250b8a |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-21T18:08:58Z |
| publishDate | 2020-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-e2f6f414774945e7a023b8eb9c250b8a2022-12-21T18:54:51ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2020-04-011110.3389/fmicb.2020.00742536030Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic AnhydraseMalvina Vogler0Malvina Vogler1Ram Karan2Dominik Renn3Alexandra Vancea4Marie-Theres Vielberg5Stefan W. Grötzinger6Priya DasSarma7Shiladitya DasSarma8Jörg Eppinger9Michael Groll10Magnus Rueping11KAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi ArabiaCenter for Integrated Protein Science Munich, Department of Chemistry, Technische Universität München, Garching, GermanyKAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi ArabiaKAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi ArabiaKAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi ArabiaCenter for Integrated Protein Science Munich, Department of Chemistry, Technische Universität München, Garching, GermanyKAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi ArabiaDepartment of Microbiology and Immunology, Institute of Marine and Environmental Technology, University of Maryland School of Medicine, Baltimore, MD, United StatesDepartment of Microbiology and Immunology, Institute of Marine and Environmental Technology, University of Maryland School of Medicine, Baltimore, MD, United StatesKAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi ArabiaCenter for Integrated Protein Science Munich, Department of Chemistry, Technische Universität München, Garching, GermanyKAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi ArabiaEnvironments 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.https://www.frontiersin.org/article/10.3389/fmicb.2020.00742/fullextremophileshalophilesthermophilesextremozymesalt adaptationmutagenesis |
| spellingShingle | Malvina Vogler Malvina Vogler Ram Karan Dominik Renn Alexandra Vancea Marie-Theres Vielberg Stefan W. Grötzinger Priya DasSarma Shiladitya DasSarma Jörg Eppinger Michael Groll Magnus Rueping Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase Frontiers in Microbiology extremophiles halophiles thermophiles extremozyme salt adaptation mutagenesis |
| title | Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase |
| title_full | Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase |
| title_fullStr | Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase |
| title_full_unstemmed | Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase |
| title_short | Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase |
| title_sort | crystal structure and active site engineering of a halophilic γ carbonic anhydrase |
| topic | extremophiles halophiles thermophiles extremozyme salt adaptation mutagenesis |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2020.00742/full |
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