Are seven amino acid substitutions sufﬁcient to explain the evolution of high L-DOPA 4,5-dioxygenase activity leading to betalain pigmentation? Revisiting the gain-of-function mutants of Bean et al. (2018)

Guerrero-Rubio, M. Alejandra; Walker-Hale, Nathanael; Guo, Rui; Sheehan, Hester; Timoneda, Alfonso; Gandía Herrero, Fernando; Brockington, Samuel F.

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Are seven amino acid substitutions sufﬁcient to explain the evolution of high L-DOPA 4,5-dioxygenase activity leading to betalain pigmentation? Revisiting the gain-of-function mutants of Bean et al. (2018)

dc.contributor.author	Guerrero-Rubio, M. Alejandra
dc.contributor.author	Walker-Hale, Nathanael
dc.contributor.author	Guo, Rui
dc.contributor.author	Sheehan, Hester
dc.contributor.author	Timoneda, Alfonso
dc.contributor.author	Gandía Herrero, Fernando
dc.contributor.author	Brockington, Samuel F.
dc.contributor.department	Bioquímica y Biología Molecular A	es
dc.coverage.temporal	2023	es
dc.date.accessioned	2025-05-09T07:04:14Z
dc.date.available	2025-05-09T07:04:14Z
dc.date.issued	2023-05-27
dc.description	© 2023 The Authors. This manuscript version is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by/4.0/. This document is the Published version of a Published Work that appeared in final form in New Phytologist. To access the final edited and published work see https://doi.org/doi.org/10.1111/nph.18981
dc.description.abstract	This work revisits a publication by Bean et al. (2018) that reports seven amino acid substitu-tions are essential for the evolution of L-DOPA 4,5-dioxygenase (DODA) activity in Caryo-phyllales. In this study, we explore several concerns which led us to replicate the analyses of Bean et al. (2018). Our comparative analyses, with structural modelling, implicate numerous residues addi-tional to those identiﬁed by Bean et al. (2018), with many of these additional residues occur-ring around the active site of BvDODAα1. We therefore replicated the analyses of Bean et al.(2018) to re-observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2-mut3 variant. Multiple in vivo assays, in both Saccharomyces cerevisiae and Nicotiana benthamiana,did not result in visible DODA activity in BvDODAα2-mut3, with betalain production always10-fold below BvDODAα1. In vitro assays also revealed substantial differences in both cataly-tic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2-mut3 proteins,explaining their differing performance in vivo. In summary, we were unable to replicate the in vivo analyses of Bean et al. (2018), and our quantitative in vivo and in vitro analyses suggest a minimal effect of these seven residues inaltering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Bean et al. (2018)	es
dc.format	application/pdf	es
dc.format.extent	12	es
dc.identifier.doi	https://doi.org/10.1111/nph.18981
dc.identifier.eisbn	New Phytologist (2023) 239: 2265–2276	es
dc.identifier.issn	Print: 0028-646X
dc.identifier.issn	Electronic: 1469-8137
dc.identifier.uri	http://hdl.handle.net/10201/154279
dc.language	eng	es
dc.publisher	Wiley
dc.relation	SFB, BBSRC High Value Chemicals from Plants Network & NERC-NSF-DEB RG88096; HS, SNF P2BEP3_165359 & P300PA_174333; NWH, Woolf Fisher Cambridge Scholarship; RG, CSC no. (2018) 3101; MAGR, European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no. 101030560.	es
dc.relation.publisherversion	https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.18981
dc.rights	info:eu-repo/semantics/openAccess	es
dc.rights	Atribución 4.0 Internacional	*
dc.rights.uri	http://creativecommons.org/licenses/by/4.0/	*
dc.subject	Betalains	es
dc.subject	Caryophyllales	es
dc.subject	DODA	es
dc.subject	Horizontal swapping	es
dc.subject	L-DOPA4	es
dc.subject	4,5-dioxygenase	es
dc.subject	Specialised metabolism	es
dc.subject.other	CDU::5 - Ciencias puras y naturales::57 - Biología::577 - Bioquímica. Biología molecular. Biofísica	es
dc.title	Are seven amino acid substitutions sufﬁcient to explain the evolution of high L-DOPA 4,5-dioxygenase activity leading to betalain pigmentation? Revisiting the gain-of-function mutants of Bean et al. (2018)	es
dc.type	info:eu-repo/semantics/article	es
dspace.entity.type	Publication	es

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