A Root-Expressed <span class="small-caps">l</span>-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in <em>Atropa belladonna</em><sup><a href="#fn1" rid="fn1" class=" fn">[C]</a></sup><sup><a href="#fn2" rid="fn2" class=" fn">[W]</a></sup>
Supplementary Material
Abstract
The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.
PF, purity filtered reads; M, million reads; nt, nucleotides.
Data are presented as the mean ± sd of three replicates.
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Funding was provided by National Institutes of General Medical Sciences Award 1RC2GM092521 to J.C., D.D.-P., C.R.B., A.D.J., and C.S.B., a Michigan State University (MSU) Foundation Strategic Partnership Grant to C.S.B. and C.R.B., and by National Science Foundation Award IOS-1025636 to C.S.B. and A.D.J. D.D.-P., C.R.B., A.D.J., and C.S.B. are supported in part by Michigan AgBioResearch and through USDA National Institute of Food and Agriculture, Hatch project numbers MICL02265 and MICL02143. M.A.B. was supported by a Summer Undergraduate Research Fellowship from the American Society of Plant Biologists, the MSU College of Agriculture and Natural Resources Undergraduate Research Program, and a fellowship from the MSU Plant Breeding, Genetics, and Biotechnology Graduate Program. The Quattro Premier mass spectrometer was purchased with funds from National Science Foundation Major Research Instrumentation Grant DBI-0619489. We thank Shari Tjugum-Holland (Research Technology Support Facility, MSU) for assistance with cDNA library preparation and operation of the Illumina Genome Analyzer IIx instrument and Scott E. Kinison (University of Kentucky) for technical assistance.
AUTHOR CONTRIBUTIONS
C.S.B., C.R.B., A.D.J., D.D.-P., and J.C. conceived and designed the research. M.A.B., E.G.-C., K.E.W.-R., E.G.-V., J.B.U., B.V., Y.-S.Y., and C.S.B. designed and performed research. M.A.B., E.G.-C., and C.S.B. analyzed the data. E.G.-C., K.L.C., and J.P.H. developed the bioinformatics pipeline. A.D.J. established mass spectrometry methods. M.A.B., E.G.-C., C.R.B., and C.S.B. wrote the article. All authors read and approved the article.
Notes
Glossary
| RNA-seq | RNA sequencing |
| LC-MS/MS | liquid chromatography-tandem mass spectrometry |
| VIGS | virus-induced gene silencing |
| TRV | tobacco rattle virus |
| PE | paired-end |
| SE | single-end |
| 4-HPP | 4-hydroxyphenylpyruvate |
| GO | Gene Ontology |
| PLP | pyridoxal 5′-phosphate |
Glossary
| RNA-seq | RNA sequencing |
| LC-MS/MS | liquid chromatography-tandem mass spectrometry |
| VIGS | virus-induced gene silencing |
| TRV | tobacco rattle virus |
| PE | paired-end |
| SE | single-end |
| 4-HPP | 4-hydroxyphenylpyruvate |
| GO | Gene Ontology |
| PLP | pyridoxal 5′-phosphate |
Footnotes
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