title: Production of (15)N-labeled α-amanitin in Galerina marginata.
abstract: α-Amanitin is the major causal constituent of deadly Amanita mushrooms that account for the majority of fatal mushroom poisonings worldwide. It is also an important biochemical tool for the study of its target, RNA polymerase II. The commercial supply of this bicyclic peptide comes from Amanita phalloides, the death cap mushroom, which is collected from the wild. Isotopically labeled amanitin could be useful for clinical and forensic applications, but α-amanitin has not been chemically synthesized and A. phalloides cannot be cultured on artificial medium. Using Galerina marginata, an unrelated saprotrophic mushroom that grows and produces α-amanitin in culture, we describe a method for producing (15)N-labeled α-amanitin using growth media containing (15)N as sole nitrogen source. A key to success was preparing (15)N-enriched yeast extract via a novel method designated "glass bead-assisted maturation." In the presence of the labeled yeast extract and (15)N-NH4Cl, α-amanitin was produced with >97% isotope enrichment. The labeled product was confirmed by HPLC, high-resolution mass spectrometry, and NMR.
authors.affiliation: Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA; Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China. Electronic address: hongluo@msu.edu.
authors.forename: Hong
authors.initials: H
authors.lastname: Luo
authors.affiliation: Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Brandon
authors.initials: B
authors.lastname: DuBois
authors.affiliation: Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA; Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: R Michael
authors.initials: RM
authors.lastname: Sgambelluri
authors.affiliation: Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Evan R
authors.initials: ER
authors.lastname: Angelos
authors.affiliation: Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA; Department of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming 650091, Yunnan, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Holmes
authors.affiliation: Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA. Electronic address: walton@msu.edu.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Walton
mesh_terms: D000363:Agaricales / Q000378:metabolism*; D053959:Alpha-Amanitin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D009682:Magnetic Resonance Spectroscopy; D009145:Mushroom Poisoning / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000737:chemistry*
chemical_list: D053959:Alpha-Amanitin; D009587:Nitrogen Isotopes
keywords: Amanita; Galerina; Isotope labeling; LC-MS; NMR; α-Amanitin