Abstract:
Citronellol is a pleasant-smelling compound produced in rose (Rosa spp.) flowers and in the leaves of many aromatic plants, including pelargoniums (Pelargonium spp.). Although geraniol production has been well studied in several plants, citronellol biosynthesis has been documented only in crab-lipped spider orchid (Caladenia plicata) and its mechanism remains open to question in other species. We therefore profiled 10 pelargonium accessions using RNA sequencing and gas chromatography-MS analysis. Three enzymes from the progesterone 5β-reductase and/or iridoid synthase-like enzymes (PRISE) family were characterized in vitroand subsequently identified as citral reductases (named PhCIRs). Transgenic RNAi lines supported a role for PhCIRs in the biosynthesis of citronellol as well as in the production of mint-scented terpenes. Despite their high amino acid sequence identity, the 3 enzymes showed contrasting stereoselectivity, either producing mainly (S)-citronellal or a racemate of both (R)- and (S)-citronellal. Using site-directed mutagenesis, we identified a single amino acid substitution as being primarily responsible for the enzyme\'s enantioselectivity. Phylogenetic analysis of pelargonium PRISEs revealed 3 clades and 7 groups of orthologs. PRISEs from different groups exhibited differential affinities toward substrates (citral and progesterone) and cofactors (NADH/NADPH), but most were able to reduce both substrates, prompting hypotheses regarding the evolutionary history of PhCIRs. Our results demonstrate that pelargoniums evolved citronellol biosynthesis independently through a 3-step pathway involving PRISE homologs and both citral and citronellal as intermediates. In addition, these enzymes control the enantiomeric ratio of citronellol thanks to small alterations of the catalytic site.
摘要:
香茅醇是玫瑰中产生的一种令人愉悦的气味化合物(Rosaspp。)花朵和许多芳香植物的叶子中,包括天花(天花属。).虽然香叶醇的生产已经在几个工厂进行了很好的研究,仅在蟹状蜘蛛兰花(Caladeniaplicata)中记录了香茅醇的生物合成,其机制在其他物种中仍存在疑问。因此,我们使用RNA-Seq和GC-MS分析对10个天竺葵种质进行了分析。来自PRISE家族的三种酶在体外进行了表征,随后被鉴定为柠檬醛还原酶(称为PhCIR)。转基因RNAi系支持PhCIR在香茅醇的生物合成以及薄荷味萜烯的生产中的作用。尽管它们具有很高的氨基酸序列同一性,这三种酶表现出相反的立体选择性,主要产生(S)-香茅醛或(R)-和(S)-香茅醛的外消旋体。使用定点诱变,我们确定了一个单一的氨基酸取代是主要负责酶的对映选择性。天盆PRISE的系统发育分析揭示了三个分支和七个直系同源物。来自不同组的PRISE对底物(柠檬醛和孕酮)和辅因子(NADH/NADPH)表现出不同的亲和力,但是大多数能够减少两种底物,提示有关PhCIR进化史的假设。我们的结果表明,天牛通过三步途径独立地进化了香茅醇的生物合成,该途径涉及PRISE同源物以及柠檬醛和香茅醛作为中间体。此外,由于催化位点的微小改变,这些酶控制香茅醇的对映体比例。