Parallel evolution of UbiA superfamily proteins into aromaticO-prenyltransferases in plants

Abstract

<jats:title>Abstract</jats:title><jats:p>Plants produce approximately 300 aromatic molecules enzymatically linked to prenyl side chains via C-O bonds. These <jats:italic>O</jats:italic>-prenylated aromatics have been found in taxonomically distant plant taxa as compounds beneficial or detrimental to human health, with <jats:italic>O</jats:italic>-prenyl moieties often playing crucial roles in their biological activities. To date, however, no plant gene encoding an aromatic <jats:italic>O</jats:italic>-prenyltransferase (<jats:italic>O</jats:italic>-PT) has been described. This study describes the isolation of an aromatic <jats:italic>O</jats:italic>-PT gene, <jats:italic>CpPT1</jats:italic>, belonging to the UbiA superfamily, from grapefruit (<jats:italic>Citrus</jats:italic> × <jats:italic>paradisi,</jats:italic> Rutaceae). This gene is responsible for the biosynthesis of <jats:italic>O</jats:italic>-prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarin <jats:italic>O</jats:italic>-PT gene of the same protein family was identified in <jats:italic>Angelica keiskei</jats:italic>, an apiaceous medicinal plant containing pharmaceutically active <jats:italic>O</jats:italic>-prenylated coumarins. Phylogenetic analysis of these <jats:italic>O</jats:italic>-PTs suggested that aromatic <jats:italic>O</jats:italic>-prenylation activity evolved independently from the same ancestral gene in these distant plant taxa. These findings shed light on understanding the evolution of plant secondary metabolites via the UbiA superfamily.</jats:p>

Characterization of a coumarin C-/O-prenyltransferase and a quinolone C-prenyltransferase from Murraya exotica

“…For the first round of screening, the candidate aromatic acceptors (1-20) were individually co-cultured with the recombinant S. cerevisiae strains correspondingly harbouring the empty pESC-HIS vector (negative control), MePT1, ΔTP-MePT1, MePT2, and ΔTP-MePT2, the metabolites in the yeast cells and culture media were extracted and carefully analysed by LC-MS. The feeding experiments revealed that MePT1 could only utilize the coumarins 1 and 2, among the twenty selected substrates (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), to generate unknown products with a molecular weight increased 136 Daltons which corresponds to the molecular weight of a geranyl chain. Surprisingly, MePT2 could not accept coumarins (1-8) as prenyl acceptors, even though the phylogenic analysis (Fig.…”

“…To analyse the catalytic potential of the candidate prenyltransferases, the recombinant yeast strains harboring the individual candidate gene were grown with 1 mM MgCl 2 and 0.1 mM aromatic acceptors (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) in synthetic galactose media lacking histidine (SG-His) for 48 h (30 °C, 200 rpm). The cultures and cells were extracted by EtOAc (containing 10% methanol), and the organic layer was combined and concentrated under vacuum.…”

“…[1][2][3] The condensation reaction is divalent metal ion-dependent and supposedly involves the formation of a strongly electrophilic prenyl carbocation which could be subsequently attacked by the electron rich aromatic ring or the phenolic hydroxy group of the aromatic acceptor, resulting in the generation of structurally diverse C-prenylated or O-prenylated products. 3,4 The differences in prenylated positions, prenyl chain lengths, and further modifications of prenyl chains greatly contribute to the extreme diversity of natural products. Huge numbers of prenylated aromatic compounds have hitherto been reported from natural plants, in particular from the Leguminosae, Moraceae, Rutaceae, and Apiaceae plants, [5][6][7][8] which resulted in the discovery of many pharmaceutically important compounds, such as the analgesic and anxiolytic agent cannabidiol from Canabis sativa, 9 the anti-cancer agent icaritin from Epimedium brevicornu, 10 and the well-known antioxidant tocopherols from higher plants.…”

“…11 Given the prenylation may increase the lipophilicity and membrane permeability of the target compounds and thus greatly improves their druggability, 12 great attention has been focused on the characterization of plant aromatic PTs for further metabolic engineering and combinatorial synthesis of medicinally important molecules, resulting in the characterization of a considerable number of C-PTs specifically employing flavonoids, resveratrols, phloroglucinols, xanthones, and coumarins as acceptors from the Leguminosae, Moraceae, Rutaceae, and Apiaceae plants. 3 In contrast, there are only two O-PTs have so far been characterized from Citrus x paradisi and Angelica keiskei, 4 although O-prenylated aromatic compounds have been extensively characterized from several medicinal plants. 13 However, neither PTs with the dual-function catalysing the Cand O-prenylation of aromatic acceptors nor PTs regio-specifically catalysing the prenylation of quinolones have been so far reported from plants.…”