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Rationally Guided Improvement of NOV1 Dioxygenase for the Conversion of Lignin-Derived Isoeugenol to Vanillin

2023-01-17Artigo científico Acesso aberto
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dc.contributor.authorDe Simone, Mario
dc.contributor.authorAlvigini, Laura
dc.contributor.authorAlonso-Cotchico, Lur
dc.contributor.authorBrissos, Vânia
dc.contributor.authorCaroli, Jonatan
dc.contributor.authorLucas, Maria Fátima
dc.contributor.authorMonza, Emanuele
dc.contributor.authorMelo, Eduardo Pinho
dc.contributor.authorMattevi, Andrea
dc.contributor.authorMartins, Lígia O.
dc.contributor.institutionInstituto de Tecnologia Química e Biológica António Xavier (ITQB)
dc.contributor.pblACS - American Chemical Society
dc.date.accessioned2023-09-30T22:19:28Z
dc.date.available2023-09-30T22:19:28Z
dc.date.issued2023-01-17
dc.descriptionFunding Information: This project has received funding from the Biobased Industries Joint Undertaking (JU) under Grant Agreement 837890 (SMARTBOX) and from Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) Grant 824017 (B-Ligzymes). The JU receives support from the European Union’s Horizon 2020 research and innovation program and the Biobased Industries Consortium (BIC). Fundação para a Ciência e a Tecnologia (FCT) additionally supported this work through R&D Unit MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020), LS4FUTURE Associated Laboratory (LA/P/0087/2020), and Project UID/Multi/04326/2019, from the operational programs CRESC Algarve 2020 and COMPETE for 2020 through Project EMBRC.PT ALG-01-0145-FEDER-022121. M.D.S. acknowledges a Ph.D. grant (2020.08246.BD) from FCT. Publisher Copyright: © 2022 American Chemical Society.
dc.description.abstractBiocatalysis is a key tool in both green chemistry and biorefinery fields. NOV1 is a dioxygenase that catalyzes the one-step, coenzyme-free oxidation of isoeugenol into vanillin and holds enormous biotechnological potential for the complete valorization of lignin as a sustainable starting material for biobased chemicals, polymers, and materials. This study integrates computational, kinetic, structural, and biophysical approaches to characterize a new NOV1 variant featuring improved activity and stability compared to those of the wild type. The S283F replacement results in a 2-fold increased turnover rate (kcat) for isoeugenol and a 4-fold higher catalytic efficiency (kcat/Km) for molecular oxygen compared to those of the wild type. Furthermore, the variant exhibits a half-life that is 20-fold higher than that of the wild type, which most likely relates to the enhanced stabilization of the iron cofactor in the active site. Molecular dynamics supports this view, revealing that the S283F replacement decreases the optimal pKa and favors conformations of the iron-coordinating histidines compatible with an increased level of binding to iron. Importantly, whole cells containing the S283F variant catalyze the conversion of ≤100 mM isoeugenol to vanillin, yielding >99% molar conversion yields within 24 h. This integrative strategy provided a new enzyme for biotechnological applications and mechanistic insights that will facilitate the future design of robust and efficient biocatalysts.en
dc.description.versionpublishersversion
dc.description.versionpublished
dc.format.extent10
dc.format.extent2358729
dc.identifier.doi10.1021/acs.biochem.2c00168
dc.identifier.issn0006-2960
dc.identifier.otherPURE: 72429487
dc.identifier.otherPURE UUID: e5ad250e-a82c-4e7e-b17a-26c9fb6e6bb5
dc.identifier.otherScopus: 85133535217
dc.identifier.otherPubMed: 35687874
dc.identifier.urihttp://hdl.handle.net/10362/158531
dc.identifier.urlhttps://www.scopus.com/pages/publications/85133535217
dc.language.isoeng
dc.peerreviewedyes
dc.subjectBiochemistry
dc.titleRationally Guided Improvement of NOV1 Dioxygenase for the Conversion of Lignin-Derived Isoeugenol to Vanillinen
dc.typejournal article
degois.publication.firstPage419
degois.publication.issue2
degois.publication.lastPage428
degois.publication.titleBiochemistry
degois.publication.volume62
dspace.entity.typePublication
rcaap.rightsopenAccess

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