Identification of pennogenin synthases in Paris polyphylla reveals enzyme plasticity underlying paralog evolution.

While artificially generated mutants offer valuable insights into protein evolutionary trajectories in laboratory settings, plasticity in paralog evolution during natural evolutionary processes remains poorly explored. In this study, we identified PpCYP81BY1 as the missing pennogenin synthase in Paris polyphylla and completed the heterologous reconstitution of multiple pharmaceutically active paris saponins in Nicotiana benthamiana. The serendipitous discovery of multiple highly similar paralogs of PpCYP81BY1 suggests recent gene-duplication events, providing an interesting case to investigate the functional fates of enzymes following gene duplication in plant metabolic evolution. Structural-guided modeling and functional analyses revealed the divergent fates of these highly similar paralogs: whereby a single-nucleotide substitution in one paralog abolishes synthase activity, while a secondary mutation in another paralog restores the activity through epistatic effects. Thus, the functional fate of paralogous enzymes in natural evolution can be switched through minimal genetic changes. Given that pennogenin synthase has also been identified in Ypsilandra thibetica, belonging to a genus that diverged from Paris prior to the massive genome expansions that arose in Paris species, their common ancestor likely encoded this enzyme during the evolution of Melanthiaceae. Taken together, our findings likely capture the relatively early-stage evolution of paralogs in P. polyphylla. The functional fate switching could be explained by a single-nucleotide substitution, which preceded subsequent mutations that may obscure a mechanistic understanding of these changes, thereby suggestive of possible evolutionary reversibility. Combined with previous findings, we propose a framework for understanding paralog evolution across life.