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)
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)
复制标题
七个氨基酸取代是否足以解释导致甜菜红素色素沉着的高 l -多巴 4,5 - 双加氧酶活性的演变？
DOI：
10.1111/nph.18981
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发表时间：
2023
期刊：
影响因子：
9.4
通讯作者：
Brockington, Samuel F.
中科院分区：
文献类型：
--
作者：
Guerrero‐Rubio, M. Alejandra;Walker‐Hale, Nathanael;Guo, Rui;Sheehan, Hester;Timoneda, Alfonso;Gandia‐Herrero, Fernando;Brockington, Samuel F.
本研究回顾了Beanet等人（2018）发表的一篇文章，该文章报道了石蕊类植物中l - DOPA 4,5 -双加氧酶（DODA）活性进化所必需的7个氨基酸替换。在本研究中，我们探讨了导致我们复制Beanet al.（2018）分析的几个问题。我们通过结构建模进行比较分析，发现除了Beanet等人（2018）发现的残基外，还有许多残基存在于BvDODAα1的活性位点周围。因此，我们重复了Beanet等人（2018）的分析，重新观察了他们在BvDODAα2背景下（即BvDODAα2‐mut3变体）原始七个残基替换的效果。在酿酒酵母和benthamiana的多次体内实验中，BvDODAα2‐mut3没有明显的DODA活性，β素的产量总是比BvDODAα1低10倍。体外实验还显示，BvDODAα1、BvDODAα2和BvDODAα2‐mut3蛋白在催化活性和pH最佳值方面存在显著差异，这解释了它们在体内的不同表现。总之，我们无法重复Beanet等人（2018）的体内分析，我们的定量体内和体外分析表明，这7个残基对改变BvDODAα2的催化活性的影响很小。我们得出结论，高DODA活性的进化途径比Beanet等人（2018）所暗示的要复杂得多。
This work revisits a publication by Beanet al.(2018) that reports seven amino acid substitutions are essential for the evolution ofl‐DOPA 4,5‐dioxygenase (DODA) activity in Caryophyllales. In this study, we explore several concerns which led us to replicate the analyses of Beanet al.(2018).Our comparative analyses, with structural modelling, implicate numerous residues additional to those identified by Beanet al.(2018), with many of these additional residues occurring around the active site of BvDODAα1. We therefore replicated the analyses of Beanet al.(2018) to re‐observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2‐mut3 variant.Multiplein vivoassays, in bothSaccharomyces cerevisiaeandNicotiana benthamiana, did not result in visible DODA activity in BvDODAα2‐mut3, with betalain production always 10‐fold below BvDODAα1.In vitroassays also revealed substantial differences in both catalytic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2‐mut3 proteins, explaining their differing performancein vivo.In summary, we were unable to replicate thein vivoanalyses of Beanet al.(2018), and our quantitativein vivoandin vitroanalyses suggest a minimal effect of these seven residues in altering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Beanet al.(2018).