Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products

A structure‐guided engineering of fructose‐6‐phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, i.e., unsubstituted alkanones and alkanals. A "smart" combinatorial library was created targeting residues D6, T26 and N28 that form a binding po...

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Detalles Bibliográficos
Autores: Junker, Sebastian, Roldán, Raquel, Joosten, Henk-Jan, Clapés Saborit, Pere, Fessner, Wolf Dieter
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2018
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/167063
Acceso en línea:http://hdl.handle.net/10261/167063
Access Level:acceso abierto
Palabra clave:Biocatalysis
Fructose-6-phosphate aldolase
HPTLC screening
Protein Engineering
Stereoselectivity
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spelling Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol productsJunker, SebastianRoldán, RaquelJoosten, Henk-JanClapés Saborit, PereFessner, Wolf DieterBiocatalysisFructose-6-phosphate aldolaseHPTLC screeningProtein EngineeringStereoselectivityA structure‐guided engineering of fructose‐6‐phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, i.e., unsubstituted alkanones and alkanals. A "smart" combinatorial library was created targeting residues D6, T26 and N28 that form a binding pocket around the nucleophilic carbon atom. Double‐selectivity screening was executed by high‐performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. While any mutation of N28 resulted in inactivation of the enzyme, D6 turned out to be the key residue that enabled activity with non‐hydroxylated nucleophiles. Altogether 25 single‐ and double‐site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6‐phosphate.This project received funding from the European Union’s Horizon 2020 research and innovation program under grant no. 635595 (CarbaZymes), the Spanish Ministerio de Economía y Competitividad (MINECO), and the Fondo Europeo de Desarrollo Regional (FEDER) (grant no. CTQ2015-63563-R to P.C.).Peer reviewedWiley-VCHMinisterio de Economía y Competitividad (España)European CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201820182018info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/167063reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/EC/H2020/635595info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/CTQ2015-63563-Rhttps://doi.org/10.1002/anie.201804831Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1670632026-05-22T06:33:51Z
dc.title.none.fl_str_mv Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products
title Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products
spellingShingle Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products
Junker, Sebastian
Biocatalysis
Fructose-6-phosphate aldolase
HPTLC screening
Protein Engineering
Stereoselectivity
title_short Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products
title_full Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products
title_fullStr Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products
title_full_unstemmed Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products
title_sort Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products
dc.creator.none.fl_str_mv Junker, Sebastian
Roldán, Raquel
Joosten, Henk-Jan
Clapés Saborit, Pere
Fessner, Wolf Dieter
author Junker, Sebastian
author_facet Junker, Sebastian
Roldán, Raquel
Joosten, Henk-Jan
Clapés Saborit, Pere
Fessner, Wolf Dieter
author_role author
author2 Roldán, Raquel
Joosten, Henk-Jan
Clapés Saborit, Pere
Fessner, Wolf Dieter
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Biocatalysis
Fructose-6-phosphate aldolase
HPTLC screening
Protein Engineering
Stereoselectivity
topic Biocatalysis
Fructose-6-phosphate aldolase
HPTLC screening
Protein Engineering
Stereoselectivity
description A structure‐guided engineering of fructose‐6‐phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, i.e., unsubstituted alkanones and alkanals. A "smart" combinatorial library was created targeting residues D6, T26 and N28 that form a binding pocket around the nucleophilic carbon atom. Double‐selectivity screening was executed by high‐performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. While any mutation of N28 resulted in inactivation of the enzyme, D6 turned out to be the key residue that enabled activity with non‐hydroxylated nucleophiles. Altogether 25 single‐ and double‐site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6‐phosphate.
publishDate 2018
dc.date.none.fl_str_mv 2018
2018
2018
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/167063
url http://hdl.handle.net/10261/167063
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/EC/H2020/635595
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/CTQ2015-63563-R
https://doi.org/10.1002/anie.201804831
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Wiley-VCH
publisher.none.fl_str_mv Wiley-VCH
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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