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Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering

Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform...

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Autores principales: Abdallah, Ingy I., van Merkerk, Ronald, Klumpenaar, Esmée, Quax, Wim J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028579/
https://www.ncbi.nlm.nih.gov/pubmed/29967474
http://dx.doi.org/10.1038/s41598-018-28177-4
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author Abdallah, Ingy I.
van Merkerk, Ronald
Klumpenaar, Esmée
Quax, Wim J.
author_facet Abdallah, Ingy I.
van Merkerk, Ronald
Klumpenaar, Esmée
Quax, Wim J.
author_sort Abdallah, Ingy I.
collection PubMed
description Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved k(cat) by 5 times. This variant can be used to enhance amorphadiene production and in turn artemisinin biosynthesis. Our findings provide the basis for the first step in improving industrial production of artemisinin and they open up possibilities for further engineering and understanding of ADS.
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spelling pubmed-60285792018-07-09 Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering Abdallah, Ingy I. van Merkerk, Ronald Klumpenaar, Esmée Quax, Wim J. Sci Rep Article Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved k(cat) by 5 times. This variant can be used to enhance amorphadiene production and in turn artemisinin biosynthesis. Our findings provide the basis for the first step in improving industrial production of artemisinin and they open up possibilities for further engineering and understanding of ADS. Nature Publishing Group UK 2018-07-02 /pmc/articles/PMC6028579/ /pubmed/29967474 http://dx.doi.org/10.1038/s41598-018-28177-4 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Abdallah, Ingy I.
van Merkerk, Ronald
Klumpenaar, Esmée
Quax, Wim J.
Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering
title Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering
title_full Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering
title_fullStr Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering
title_full_unstemmed Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering
title_short Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering
title_sort catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028579/
https://www.ncbi.nlm.nih.gov/pubmed/29967474
http://dx.doi.org/10.1038/s41598-018-28177-4
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