Tracing the stepwise Darwinian evolution of a plant halogenase

Abstract

Biohalogenation is rare in plant metabolism, with the Menispermaceae’s chloroalkaloid acutumine being an exception. This involves a specialized dechloroacutumine halogenase (DAH) from the iron- and 2-oxoglutarate–dependent dioxygenase (2ODD) family. While DAH is presumed to have evolved from an ancestral 2ODD, how enzyme specialization arises through Darwinian processes remains a fundamental question in understanding metabolic evolution. Here, we investigate the evolutionary history of DAH using the chromosomal-level genome of Menispermum canadense. Phylogenomic dating and synteny analyses reveal DAH evolution through tandem duplication of an ancestral flavonol synthase (FLS) gene, followed by neofunctionalization and gene loss events. Structural modeling, molecular dynamics, and site-directed mutagenesis identify mutations enabling the catalytic switch from FLS to DAH. This required traversing a complex evolutionary landscape with deep fitness valleys separating intermediate states captured in the M. canadense genome. Our findings illustrate how enzymatic functions evolve through lineage-specific pathways, reshaping active sites and enabling catalytic mechanism-switching mutations.