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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)
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)
Summary

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).

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Award ID(s):
1939226
PAR ID:
10492353
Author(s) / Creator(s):
; ; ; ; ; ;
Publisher / Repository:
Wiley
Date Published:
Journal Name:
New Phytologist
Volume:
239
Issue:
6
ISSN:
0028-646X
Page Range / eLocation ID:
2265 to 2276
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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