Browsing by Subject "Caryophyllales"

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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)
    (Wiley, 2023-05-27) Guerrero-Rubio, M. Alejandra; Walker-Hale, Nathanael; Guo, Rui; Sheehan, Hester; Timoneda, Alfonso; Gandía Herrero, Fernando; Brockington, Samuel F.; Bioquímica y Biología Molecular A
    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)
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    Lineage-specific gene radiations underlie the evolution of novel betalain pigmentation in Caryophyllales
    (Wiley, 2015-05-13) Brockington, Samuel F.; Yang, Ya; Gandía Herrero, Fernando; Covshoff, Sarah; Hibberd, Julián M.; Sage, Rowan F.; Wong, Gane K. S.; Moore, Michael J.; Smith, Stephen A.; Bioquímica y Biología Molecular "A"
    Betalain pigments are unique to the Caryophyllales and structurally and biosynthetically dis-tinct from anthocyanins. Two key enzymes within the betalain synthesis pathway have beenidentified: 4,5-dioxygenase (DODA) that catalyzes the formation of betalamic acid andCYP76AD1, a cytochrome P450 gene that catalyzes the formation of cyclo-DOPA. We performed phylogenetic analyses to reveal the evolutionary history of the DODA andCYP76AD1 lineages and in the context of an ancestral reconstruction of pigment states weexplored the evolution of these genes in relation to the complex evolution of pigments inCaryophylalles. Duplications within the CYP76AD1 and DODA lineages arose just before the origin of beta-lain pigmentation in the core Caryophyllales. The duplications gave rise to DODA-a andCYP76AD1-a isoforms that appear specific to betalain synthesis. Both betalain-specific isoformswere then lost or downregulated in the anthocyanic Molluginaceae and Caryophyllaceae. Our findings suggest a single origin of the betalain synthesis pathway, with neofunctionaliza-tion following gene duplications in the CYP76AD1 and DODA lineages. Loss of DODA-a andCYP76AD1-a in anthocyanic taxa suggests that betalain pigmentation has been lost twice inCaryophyllales, and exclusion of betalain pigments from anthocyanic taxa is mediated throughgene loss or downregulation. [Correction added after online publication 13 May 2015: in thelast two paragraphs of the Summary the gene name CYP761A was changed to CYP76AD1.]