title: A root-expressed L-phenylalanine:4-hydroxyphenylpyruvate aminotransferase is required for tropane alkaloid biosynthesis in Atropa belladonna.
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.
authors.affiliation: Department of Horticulture, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Bedewitz
authors.affiliation: Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Elsa
authors.initials: E
authors.lastname: Góngora-Castillo
authors.affiliation: Department of Horticulture, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Uebler
authors.affiliation: Department of Horticulture, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Eliana
authors.initials: E
authors.lastname: Gonzales-Vigil
authors.affiliation: Department of Horticulture, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Krystle E
authors.initials: KE
authors.lastname: Wiegert-Rininger
authors.affiliation: Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Childs
authors.affiliation: Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: John P
authors.initials: JP
authors.lastname: Hamilton
authors.affiliation: Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Brieanne
authors.initials: B
authors.lastname: Vaillancourt
authors.affiliation: Plant Biology Program and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40546.
authors.forename: Yun-Soo
authors.initials: YS
authors.lastname: Yeo
authors.affiliation: Plant Biology Program and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40546.
authors.forename: Joseph
authors.initials: J
authors.lastname: Chappell
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Dean
authors.initials: D
authors.lastname: DellaPenna
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 Department of Chemistry, Michigan State University, East Lansing, Michigan 48824.
authors.forename: A Daniel
authors.initials: AD
authors.lastname: Jones
authors.affiliation: Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: C Robin
authors.initials: CR
authors.lastname: Buell
authors.affiliation: Department of Horticulture, Michigan State University, East Lansing, Michigan 48824 barrycs@msu.edu.
authors.forename: Cornelius S
authors.initials: CS
authors.lastname: Barry
mesh_terms: D001532:Atropa belladonna / Q000201:enzymology* / Q000235:genetics; D053898:Biosynthetic Pathways / Q000235:genetics; D003198:Computer Simulation; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D007700:Kinetics; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D005810:Multigene Family; D010649:Phenylalanine / Q000378:metabolism*; D010667:Phenylpyruvic Acids / Q000378:metabolism*; D010802:Phylogeny; D018517:Plant Roots / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*; D059467:Transcriptome / Q000235:genetics; D014326:Tropanes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010667:Phenylpyruvic Acids; D012333:RNA, Messenger; D014326:Tropanes; C010590:4-hydroxyphenylpyruvic acid; D010649:Phenylalanine; D000637:Transaminases; C019729:aromatic amino acid aminotransferase