Molecular evolution of parsnip (Pastinaca sativa) membrane‐bound prenyltransferases for linear and/or angular furanocoumarin biosynthesis

Abstract

SummaryIn Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone.Here, we report cloning and functional characterization of two different prenyltransferases, Pastinaca sativa prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions.Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol.These results are the first example of a complementary prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin.

A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip

“…The assembled sequences of the different BAC clones (27B24 and 102B12 for CYP71AJ3‐CYP71AJ4 ; 67N6 and 112I2 for PsPT1 ) were annotated to explore their coding sequences content. We searched for gene families belonging to cytochrome P450s, Fe(II) α‐ketoglutarate‐dependent dioxygenases, prenyltransferases and methyltransferases, which have already been reported to play roles in furanocoumarin synthesis in higher plants (Hehmann et al ., ; Larbat et al ., , ; Vialart et al ., ; Munakata et al ., ). Additionally, we extended our investigation to other proteins such as transporters and transcription factors (Table S2).…”

“…PCR products corresponding to part of PsPT1 gene were sequenced to validate that the clones carried the targeted sequence. Sequencing of the corresponding PCR products revealed 100% identity with the PsPT1 sequence for three BAC clones (67N6, 92M14 and 112I2) (Munakata et al ., ). In the fourth clone (57P4), 2 SNPs (single‐nucleotide polymorphisms) were detected.…”

“…In this study we focused on P. sativa as a model plant for which four genes encoding enzymes related to furanocoumarin biosynthesis have already been reported (Figure ). Two prenyltransferases (PsPT1 and PsPT2) were recently shown to catalyze the prenylation of umbelliferone on C6 or C8, yielding demethylsuberosin (DMS) and osthenol, respectively (Munakata et al ., ). These enzymes have the same activity as an orthologous enzyme previously identified in parsley (Karamat et al ., ) but are preferentially involved in the synthesis of linear or angular isomers.…”

Molecular Characterization of Soybean Pterocarpan 2-Dimethylallyltransferase in Glyceollin Biosynthesis: Local Gene and Whole-Genome Duplications of Prenyltransferase Genes Led to the Structural Diversity of Soybean Prenylated Isoflavonoids

“…Prenylation of flavonoids is catalyzed by prenyltransferases (PTs), which transfer prenyl moieties from allylic prenyl diphosphate to flavonoid skeletons. The flavonoid-specific PT naringenin 8-dimethylallyltransferase (SfN8DT) was initially identified in the leguminous plant Sophora flavescens (Sasaki et al 2008), and several plant PTs that act on flavonoids and other aromatic compounds have been molecularly and biochemically characterized (Akashi et al 2009, Sasaki et al 2011, Shen et al 2012, Chen et al 2013, Karamat et al 2014, Li et al 2014, Munakata et al 2014, Wang et al 2014, Li et al 2015, Munakata et al 2016). A recent report on hop PTs demonstrated that a complex of two PTs, as a metabolon, catalyzes three steps of sequential aromatic prenylation in β-bitter acid biosynthesis (Li et al 2015).…”

A Distinct Aromatic Prenyltransferase Associated with the Futalosine Pathway

“…Alternatively, the absence of a cofactor or accessory protein and/or the requirement for alternative prenyl acceptor / donor substrates might explain our findings. That members of the UbiA superfamily can possess exquisite specificity is borne out by recent studies on plant secondary metabolite aromatic prenyltransferases, – for example, of 17 potential prenyl acceptor and 3 donor substrate combinations (a total of 51) tested, only umbelliferone / DMAPP were accepted by the coumarin‐specific prenyltransferase from parsley ( Pc PT) . Concerning the aromatic moiety, further modifications of 1,4‐dihydroxy‐6‐naphthoate might be necessary to generate the acceptor substrate for MqnP.…”