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

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.

[1]  D. Kliebenstein,et al.  Natural Variation of Plant Metabolism: Genetic Mechanisms, Interpretive Caveats, and Evolutionary and Mechanistic Insights1 , 2015, Plant Physiology.

[2]  Takayuki Tohge,et al.  Metabolomic Characterization of Knockout Mutants in Arabidopsis: Development of a Metabolite Profiling Database for Knockout Mutants in Arabidopsis1[W][OPEN] , 2014, Plant Physiology.

[3]  E. Birney,et al.  Pfam: the protein families database , 2013, Nucleic Acids Res..

[4]  Manhoi Hur,et al.  Identification and biosynthesis of acylphloroglucinols in Hypericum gentianoides. , 2013, Physiologia Plantarum : An International Journal for Plant Biology.

[5]  K. Olsen,et al.  A bountiful harvest: genomic insights into crop domestication phenotypes. , 2013, Annual review of plant biology.

[6]  Márcia M. Almeida-de-Macedo,et al.  A global approach to analysis and interpretation of metabolic data for plant natural product discovery. , 2013, Natural product reports.

[7]  C. Buell,et al.  Development of Transcriptomic Resources for Interrogating the Biosynthesis of Monoterpene Indole Alkaloids in Medicinal Plant Species , 2012, PloS one.

[8]  A. D. Jones,et al.  Functional Identification of Valerena-1,10-diene Synthase, a Terpene Synthase Catalyzing a Unique Chemical Cascade in the Biosynthesis of Biologically Active Sesquiterpenes in Valeriana officinalis* , 2012, The Journal of Biological Chemistry.

[9]  Narmada Thanki,et al.  CDD: conserved domains and protein three-dimensional structure , 2012, Nucleic Acids Res..

[10]  P. Dixon,et al.  Medicinal Plants: A Public Resource for Metabolomics and Hypothesis Development , 2012, Metabolites.

[11]  S. Abbo,et al.  A critical review of the protracted domestication model for Near-Eastern founder crops: linear regression, long-distance gene flow, archaeological, and archaeobotanical evidence. , 2012, Journal of experimental botany.

[12]  Anthony L. Schilmiller,et al.  Taming the hydra of specialized metabolism: how systems biology and comparative approaches are revolutionizing plant biochemistry. , 2012, Current opinion in plant biology.

[13]  M. Ríos.  Natural Alkamides: Pharmacology, Chemistry and Distribution , 2012 .

[14]  Martin Vingron,et al.  Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels , 2012, Bioinform..

[15]  Insuk Lee,et al.  Metabolomics as a Hypothesis-Generating Functional Genomics Tool for the Annotation of Arabidopsis thaliana Genes of “Unknown Function” , 2012, Front. Plant Sci..

[16]  M. Nei,et al.  MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. , 2011, Molecular biology and evolution.

[17]  A. Schieber,et al.  Analysis of alkylamides in Echinacea plant materials and dietary supplements by ultrafast liquid chromatography with diode array and mass spectrometric detection. , 2011, Journal of agricultural and food chemistry.

[18]  P. Ríos-Chávez,et al.  Valine and Phenylalanine as Precursors in the Biosynthesis of Alkamides in Acmella Radicans , 2011, Natural product communications.

[19]  Eran Pichersky,et al.  Convergent evolution in plant specialized metabolism. , 2011, Annual review of plant biology.

[20]  C. Buell,et al.  A Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in Catharanthus roseus* , 2011, The Journal of Biological Chemistry.

[21]  E. Wurtele,et al.  Reverse-Genetic Analysis of the Two Biotin-Containing Subunit Genes of the Heteromeric Acetyl-Coenzyme A Carboxylase in Arabidopsis Indicates a Unidirectional Functional Redundancy1[C][W][OA] , 2010, Plant Physiology.

[22]  Cole Trapnell,et al.  Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. , 2010, Nature biotechnology.

[23]  L. Mueller,et al.  A Dynamic Interface for Capsaicinoid Systems Biology1[C][W][OA] , 2009, Plant Physiology.

[24]  Lior Pachter,et al.  Sequence Analysis , 2020, Definitions.

[25]  P. Dixon,et al.  Metabolic profiling of echinacea genotypes and a test of alternative taxonomic treatments. , 2009, Planta medica.

[26]  Juan Carlos Campos-Cuevas,et al.  Tissue culture of Arabidopsis thaliana explants reveals a stimulatory effect of alkamides on adventitious root formation and nitric oxide accumulation , 2008 .

[27]  D. Fuller.  Contrasting Patterns in Crop Domestication and Domestication Rates: Recent Archaeobotanical Insights from the Old World , 2007, Annals of botany.

[28]  R. Carle,et al.  Determination of free amino compounds in betalainic fruits and vegetables by gas chromatography with flame ionization and mass spectrometric detection. , 2006, Journal of agricultural and food chemistry.

[29]  R. Bauer,et al.  The Endocannabinoid System as a Target for Alkamides from Echinacea angustifolia Roots , 2005, Planta medica.

[30]  Alok J. Saldanha,et al.  Java Treeview - extensible visualization of microarray data , 2004, Bioinform..

[31]  A. Suter,et al.  Echinacea alkylamides modulate TNF‐α gene expression via cannabinoid receptor CB2 and multiple signal transduction pathways , 2004, FEBS letters.

[32]  E. Wurtele,et al.  Diacetylenic isobutylamides of Echinacea: synthesis and natural distribution. , 2004, Phytochemistry.

[33]  S Miyano,et al.  Open source clustering software. , 2004, Bioinformatics.

[34]  L. Herrera-Estrella,et al.  Alkamides Isolated from Plants Promote Growth and Alter Root Development in Arabidopsis1 , 2004, Plant Physiology.

[35]  Jack F Kirsch,et al.  Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. , 2003, Annual review of biochemistry.

[36]  J. Hoyt,et al.  Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin , 2002, Molecular microbiology.

[37]  V. Cavrini,et al.  GC-MS analysis of the lipophilic principles of Echinacea purpurea and evaluation of cucumber mosaic cucumovirus infection. , 2002, Journal of pharmaceutical and biomedical analysis.

[38]  Eve Syrkin Wurtele,et al.  Metabolic and Environmental Regulation of 3-Methylcrotonyl-Coenzyme A Carboxylase Expression in Arabidopsis1 , 2002, Plant Physiology.

[39]  S. E. Binns,et al.  Phytochemical variation in echinacea from roots and flowerheads of wild and cultivated populations. , 2002, Journal of agricultural and food chemistry.

[40]  A. Hanson,et al.  Plants Synthesize Ethanolamine by Direct Decarboxylation of Serine Using a Pyridoxal Phosphate Enzyme* , 2001, The Journal of Biological Chemistry.

[41]  S. E. Binns,et al.  Methyl jasmonate increases reported alkamides and ketoalkene/ynes in Echinacea pallida (Asteraceae). , 2001, Phytochemistry.

[42]  S. Brunak,et al.  Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. , 2000, Journal of molecular biology.

[43]  C. V. Jongeneel,et al.  ESTScan: A Program for Detecting, Evaluating, and Reconstructing Potential Coding Regions in EST Sequences , 1999, ISMB.

[44]  S. Cutler,et al.  Biologically Active Natural Products: Pharmaceuticals , 1999 .

[45]  C. Olsen,et al.  Phytochemistry of the genus Piper , 1997 .

[46]  T. Namba,et al.  Amides of the fruit of Zanthoxylum spp. , 1997 .

[47]  H Hayashi,et al.  Pyridoxal enzymes: mechanistic diversity and uniformity. , 1995, Journal of biochemistry.

[48]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[49]  P. Christen,et al.  Multiple evolutionary origin of pyridoxal-5'-phosphate-dependent amino acid decarboxylases. , 1994, European journal of biochemistry.

[50]  William R. Taylor,et al.  The rapid generation of mutation data matrices from protein sequences , 1992, Comput. Appl. Biosci..

[51]  R. Bauer,et al.  Analysis of alkamides and caffeic acid derivatives from Echinacea simulata and E. paradoxa roots. , 1991, Planta medica.

[52]  M. Kanehisa,et al.  Expert system for predicting protein localization sites in gram‐negative bacteria , 1991, Proteins.

[53]  D. Stevenson,et al.  L-methionine decarboxylase from Dryopteris filix-mas: purification, characterization, substrate specificity, abortive transamination of the coenzyme, and stereochemical courses of substrate decarboxylation and coenzyme transamination. , 1990, Biochemistry.

[54]  G. L. Vaaler,et al.  Pyridoxal 5'-phosphate dependent histidine decarboxylase: overproduction, purification, biosynthesis of soluble site-directed mutant proteins, and replacement of conserved residues. , 1989, Biochemistry.

[55]  R. Bauer,et al.  TLC and HPLC Analysis of Alkamides in Echinacea Drugs1,2 , 1989, Planta medica.

[56]  N. Ohno,et al.  Insecticidal Unsaturated Isobutylamides: From Natural Products to Agrochemical Leads , 1989 .

[57]  R. Bauer,et al.  TLC and HPLC Analysis of Echinacea pallida and E. angustifolia Roots1 , 1988, Planta medica.

[58]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[59]  M. Brasch,et al.  Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Nucleotide sequence of the hdc gene and the corresponding amino acid sequence. , 1986, The Journal of biological chemistry.

[60]  M. Gilmore.  Uses of Plants by the Indians of the Missouri River Region , 1977 .

[61]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[62]  J. D. Lee,et al.  Interpretation of mass spectra. , 1973, Talanta.

[63]  C Robin Buell,et al.  Genomic approaches for interrogating the biochemistry of medicinal plant species. , 2012, Methods in enzymology.

[64]  E. Pichersky,et al.  Plant metabolism. Editorial. , 2010, Molecular plant.

[65]  J. Lam,et al.  Acetylenes and related compounds in heliantheae , 1991 .

[66]  R. Bauer,et al.  Alkamides from the roots of Echinacea angustifolia , 1989 .

[67]  R. Bauer,et al.  Alkamides from the roots of Echinacea purpurea , 1988 .

[68]  Fred W. McLafferty,et al.  Mass Spectrometric Analysis. Molecular Rearrangements , 1959 .