Convergent Biochemical Pathways for Xanthine Alkaloid Production in Plants Evolved from Ancestral Enzymes with Different Catalytic Properties

Abstract

Convergent evolution is widespread but the extent to which common ancestral conditions are necessary to facilitate the independent acquisition of similar traits remains unclear. In order to better understand how ancestral biosynthetic catalytic capabilities might lead to convergent evolution of similar modern-day biochemical pathways, we resurrected ancient enzymes of the Caffeine Synthase (CS) methyltransferases that are responsible for theobromine and caffeine production in flowering plants. Ancestral CS enzymes of Theobroma, Paullinia and Camellia exhibited similar substrate preferences but these resulted in the formation of different sets of products. From these ancestral enzymes, descendants with similar substrate preference and product formation independently evolved after gene duplication events in Theobroma and Paullinia. Thus, it appears that the convergent modern-day pathways likely originated from ancestral pathways with different inferred flux. Subsequently, the modern-day enzymes originated independently via gene duplication and their convergent catalytic characteristics evolved to partition the multiple ancestral activities by different mutations that occurred in homologous regions of the ancestral proteins. These results show that even when modern-day pathways and recruited genes are similar, the antecedent conditions may be distinctive such that different evolutionary steps are required to generate convergence.

Yerba mate (Ilex paraguariensis) genome provides new insights into convergent evolution of caffeine biosynthesis

“…7b). This homologous region was experimentally mutated in Theobroma and Paullinia CS2 enzymes and improved specialization for 3X methylation in both, although the actual substitutions are different in each case 24 . Thus, this may represent an additional instance where convergent mutations of the same region lead to specialization for 3X methylation.…”

“…In Camellia , Theobroma and Paullinia , recent duplications of the CS-type enzymes responsible for the successive steps of xanthine alkaloid methylation have also independently occurred (Fig. 3a) 19, 24 . Two other YM genes encode IpCS4 & 5, but these are not syntenic with IpCS1-3 and are not highly expressed in any tissues studied (Fig.…”

“…Not only did the modern-day CS enzymes of YM evolve to catalyse a pathway from X>3X>TB>CF from ancestral biosynthetic networks of different products, Theobroma and Paullinia also independently evolved enzymes with similar properties 19 . And, they did so from ancestral pathways that, like YM, had alternative ancestral fluxes 24 . While it could be due to chance alone that all three lineages converged to catalyse a similar pathway from differing ancestral networks, it is also possible that it was advantageous to specialize for flux to TB via X and 3X because either it is more enzymatically favourable or these intermediates have greater adaptive value than other structural isomers.…”

“…1). Convergence appears to also extend to the mutational level, since different amino acid replacements to homologous regions of CS and XMT enzymes appear to govern the evolution of substrate preference switches 24 . However, it remains unclear whether mutations lead to convergent three-dimensional protein structures to confer convergent substrate interactions and catalysis by the enzymes.…”

“…Nitrogen atoms are coloured to match the arrows corresponding to the enzymes that methylate them. Adapted with permission from O’Donnell et al 24…”