Integrating metabolomics and transcriptomics data to discover a biocatalyst that can generate the amine precursors for alkamide biosynthesis

Abstract

Summary The Echinacea genus is exemplary of over 30 plant families that produce a set of bioactive amides, called alkamides. The Echinacea alkamides may be assembled from two distinct moieties, a branched-chain amine that is acylated with a novel polyunsaturated fatty acid. In this study we identified the potential enzymological source of the amine moiety as a pyridoxal phosphate dependent decarboxylating enzyme that uses branched chain amino acids as substrate. This identification was based on a correlative analysis of the transcriptomes and metabolomes of 36 different E. purpurea tissues and organs, which expressed distinct alkamide profiles. Although no correlation was found between the accumulation patterns of the alkamides and their putative metabolic precursors (i.e., fatty acids and branched chain amino acids), isotope-labeling analyses supported the transformation of valine and isoleucine to isobutylamine and 2-methylbutylamine as reactions of alkamide biosynthesis. Sequence homology identified the pyridoxal phosphate dependent decarboxylase-like proteins in the translated proteome of E. purpurea. These sequences were prioritized for direct characterization by correlating their transcript levels with alkamide accumulation patterns in different organs and tissues, and this multi-pronged approach led to the identification and characterization of a branched-chain amino acid decarboxylase, which would appear to be responsible for generating the amine moieties of naturally occurring alkamides.

Plant-endophytes interaction influences the secondary metabolism in Echinacea purpurea (L.) Moench: an in vitro model

“…In fact, both alkamide content and VDC transcription level resulted increased after the infection. The transcriptional up-regulation in the infected R samples was lower than the expected one: this result could be explained considering that the increase of the alkamide quantity in the infected R might be due to the transcriptional regulation of other enzymes required for the biosynthesis of the alkamide ammine and fatty acid moieties 24 . Interestingly, the SDC enzyme did not seem to contribute to the increase of alkamide levels confirming the specificity of the enzymatic source for the amine moiety formation of the alkamides ( i.e .…”

“…Also, the expression level resulted increased in R infected tissues respect to the relative controls even if at a minor extent ( P < 0.01). In parallel, the involvement of an enzyme (serine decarboxylase SDC, GenBank Accession #LT593931.1) not decarboxylating valine or isoleucine (to form isobutylamine or 2-methylbutylamine, the amine moieties of the E. purpurea alkamides) but utilizing the serine as substrate to generate ethanolamine 24 was evaluated. The transcription of the SDC gene (slope SDC = −3.135; R 2 SDC = 0.982) appeared down-regulated in the infected SL samples respect to the SL control ones whilst the SDC expression levels were similar between control and infected R samples (Fig.…”

“…The primer sequences for the VDC gene were reported in Rizhsky et al . 24 . All primers listed in Supplementary Table 2 were synthesized by Eurofins Genomics (Ebersberg, Germany).…”

“…Recently, a pyridoxal phosphate-dependent (PLP) decarboxylating enzyme belonging to the Class II tryptophan synthase family that utilizes branched-chain amino acids (BCAA) as substrate has been suggested 24 . Isotope labelling analyses have revealed the generation of isobutylamine and 2-methylbutylamine ( i.e .…”

Phytochemical localization and fungal inhabitants in the fruits of four Echinacea taxa

“…Particularly high concentrations of alkylamides were found in petals and disc flowers, and moderate concentrations were also noted in receptacles of mature flower heads. However, this study did not analyze flower head or seed samples after seed set (Rizhsky et al 2016).…”