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Engineering of Ance...
Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase
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- Schriever, Karen (författare)
- KTH,Fiber- och polymerteknologi,Science for Life Laboratory, SciLifeLab,Sci Life Lab, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.;KTH Royal Inst Technol, Dept Fibre & Polymer Technol, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.,KTH Royal Institute of Technology
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- Saenz Mendez, Patricia (författare)
- KTH,Science for Life Laboratory, SciLifeLab,Fiber- och polymerteknologi,Sci Life Lab, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.;KTH Royal Inst Technol, Dept Fibre & Polymer Technol, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.,KTH Royal Institute of Technology
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- Rudraraju, Reshma Srilakshmi (författare)
- Karolinska Institutet,Karolinska Inst, Dept Med Biochem & Biophys, S-17165 Stockholm, Sweden.
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- Hendrikse, Natalie (författare)
- KTH,Ytbehandlingsteknik,Sci Life Lab, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.;KTH Royal Inst Technol, Dept Fibre & Polymer Technol, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.,KTH Royal Institute of Technology
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- Hudson, Elton P. (författare)
- KTH,Science for Life Laboratory, SciLifeLab,Sci Life Lab, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.;KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, S-11428 Stockholm, Sweden.,KTH Royal Institute of Technology
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- Biundo, Antonino (författare)
- KTH,Ytbehandlingsteknik,Sci Life Lab, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.;KTH Royal Inst Technol, Dept Fibre & Polymer Technol, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.,KTH Royal Institute of Technology
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- Schnell, Robert (författare)
- Karolinska Institutet
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- Syrén, Per-Olof (författare)
- KTH,Science for Life Laboratory, SciLifeLab,Ytbehandlingsteknik,Wallenberg Wood Science Center,Sci Life Lab, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.;KTH Royal Inst Technol, Dept Fibre & Polymer Technol, Sch Engn Sci Chem Biotechnol & Hlth, S-11428 Stockholm, Sweden.;Wallenberg Wood Sci Ctr, S-10044 Stockholm, Sweden.,KTH Royal Institute of Technology
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(creator_code:org_t)
- 2021-01-26
- 2021
- Engelska.
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 143:10, s. 3794-3807
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https://urn.kb.se/re...
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https://doi.org/10.1...
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http://kipublication...
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Abstract
Ämnesord
Stäng
- Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme's specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Nyckelord
- Chemistry - Biochemistry
- Kemi - biokemi
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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