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Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase

dc.contributor.authorBorges, Patrícia T.
dc.contributor.authorSilva, Diogo
dc.contributor.authorSilva, Tomás F.D.
dc.contributor.authorBrissos, Vânia
dc.contributor.authorCañellas, Marina
dc.contributor.authorLucas, Maria Fátima
dc.contributor.authorMasgrau, Laura
dc.contributor.authorMelo, Eduardo
dc.contributor.authorMachuqueiro, Miguel
dc.contributor.authorFrazão, Carlos
dc.contributor.authorMartins, Lígia O.
dc.date.accessioned2023-02-07T11:40:17Z
dc.date.available2023-02-07T11:40:17Z
dc.date.issued2022-12
dc.description.abstractDyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range of substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn2+ and Fe2+, and have enormous biotechnological potential in biorefineries. However, many questions on the molecular basis of enzyme function and stability remain unanswered. In this work, high-resolution structures of PpDyP wild-type and two engineered variants (6E10 and 29E4) generated by directed evolution were obtained. The X-ray crystal structures revealed the typical ferredoxin-like folds, with three heme access pathways, two tunnels, and one cavity, limited by three long loops including catalytic residues. Variant 6E10 displays significantly increased loops' flexibility that favors function over stability: despite the considerably higher catalytic efficiency, this variant shows poorer protein stability compared to wild-type and 29E4 variants. Constant-pH MD simulations revealed a more positively charged microenvironment near the heme pocket of variant 6E10, particularly in the neutral to alkaline pH range. This microenvironment affects enzyme activity by modulating the pK(a) of essential residues in the heme vicinity and should account for variant 6E10 improved activity at pH 7-8 compared to the wild-type and 29E4 that show optimal enzymatic activity close to pH 4. Our findings shed light on the structure-function relationships of DyPs at the molecular level, including their pH-dependent conformational plasticity. These are essential for understanding and engineering the catalytic properties of DyPs for future biotechnological applications. (c) 2022 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.pt_PT
dc.description.sponsorshipLA/P/0087/2020
dc.description.sponsorshipLA/P/0101/2020
dc.description.sponsorshipALG-01-0145-FEDER-022121
dc.description.sponsorshipPGC2018-098592-B-100
dc.description.sponsorshipPID2021-126897NB-100
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doi10.1016/j.csbj.2022.07.032pt_PT
dc.identifier.issn2001-0370
dc.identifier.urihttp://hdl.handle.net/10400.1/19004
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherElsevierpt_PT
dc.relationNot Available
dc.relationMolecular, Structural and Cellular Microbiology – Instituto de Tecnologia Química e Biológica António Xavier
dc.relationMolecular, Structural and Cellular Microbiology – Instituto de Tecnologia Química e Biológica António Xavier
dc.relationBiosystems and Integrative Sciences Institute
dc.relationBiosystems and Integrative Sciences Institute
dc.relationAlgarve Centre for Marine Sciences
dc.relationAlgarve Centre for Marine Sciences
dc.relationTool-box of engineered DyP-type peroxidases for lignin degradation and valorisation
dc.relationDesenvolvimento e aplicação de técnicas de amostragem aumentada dependente do pH na optimização da tecnologia pHLIP como marcador tumoral
dc.rights.urihttp://creativecommons.org/licenses/by-nd/4.0/pt_PT
dc.subjectBiorefinerypt_PT
dc.subjectBiocatalysispt_PT
dc.subjectDirected evolutionpt_PT
dc.subjectProtein stabilitypt_PT
dc.subjectStructure-function relationshipspt_PT
dc.titleUnveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidasept_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleNot Available
oaire.awardTitleMolecular, Structural and Cellular Microbiology – Instituto de Tecnologia Química e Biológica António Xavier
oaire.awardTitleMolecular, Structural and Cellular Microbiology – Instituto de Tecnologia Química e Biológica António Xavier
oaire.awardTitleBiosystems and Integrative Sciences Institute
oaire.awardTitleBiosystems and Integrative Sciences Institute
oaire.awardTitleAlgarve Centre for Marine Sciences
oaire.awardTitleAlgarve Centre for Marine Sciences
oaire.awardTitleTool-box of engineered DyP-type peroxidases for lignin degradation and valorisation
oaire.awardTitleDesenvolvimento e aplicação de técnicas de amostragem aumentada dependente do pH na optimização da tecnologia pHLIP como marcador tumoral
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/CEEC IND 2017/CEECIND%2F02300%2F2017%2FCP1387%2FCT0031/PT
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oaire.citation.endPage3910pt_PT
oaire.citation.startPage3899pt_PT
oaire.citation.titleComputational and Structural Biotechnology Journalpt_PT
oaire.citation.volume20pt_PT
oaire.fundingStreamCEEC IND 2017
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person.familyNamePinho Melo
person.givenNameEduardo
person.identifier1443188
person.identifier.ciencia-id3C1C-C10C-1510
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person.identifier.scopus-author-id35566177900
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rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT
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