Mechanisms of Enzyme Neofunctionalization in Plant Specialized Metabolism

Abstract

Plants, as sessile organisms, have evolved a plethora of secondary metabolites, along with highly specialized enzymes and unique metabolic pathways that support their biosynthesis. To date, approximately one-third of all existing pharmaceuticals are of plant origin. However, only a fraction of plant natural products and their derivatives have been explored as potential therapeutics. Moreover, the majority of plant natural product biosynthetic pathways remain inadequately studied, and the biocatalytic potential of their biosynthetic enzymes remains underexplored. In this thesis, we explore the molecular mechanisms underlying enzyme neofunctionalization in plant specialized metabolism by examining two enzyme families: nonheme iron(II)- and 2-oxoglutarate-dependent halogenase and BAHD acyltransferase. First, we describe the genomic- and biochemical-basis for gene duplication and neofunctionalization of flavonol synthase (FLS) that led to the unique emergence of halogenase in Menispermaceae plants. The discovery of dechloroacutumine halogenase (DAH) that catalyzes the regio-stereoselective chlorination in acutumine biosynthesis, exemplifies a mode of action for alkaloid diversification. Furthermore, we present the chromosomal-level assembly of Menispermum canadense genome, which provides genomic insights to plant halogenase evolution. Next, we examine the structural- and mechanistic-basis for the neofunctionalization of coumarin synthase (COSY) that catalyzes the isomerization and lactonization in coumarin biosynthesis. This study unveils the emergence of an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on its recruitment to the evolutionarily new coumarin biosynthetic pathway in eudicots. Overall, this work focuses on the remarkable strategies that plants explore to evolve their biocatalytic machinery and to expand their specialized metabolism.