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.

Keywords: Amanita; Galerina; Isotope labeling; LC-MS; NMR; α-Amanitin.

Fig. 1

Liquid culture of G. marginata. A. Shake culture. B. Close-up of flasks showing mycelial balls.

Fig. 2

Time course of ¹⁵N-labeled α-amanitin accumulation in G. marginata grown in shake culture. Each data point is the average of two biological replicates. Error bars indicate the range.

Fig. 3

HPLC chromatograms of the (A) first and (B) third separation of ¹⁵N-labeled α-amanitin. The y-axis is OD₂₈₀ (mAU).

Fig. 4

Mass spectrometric analysis of (A) native and (B) ¹⁵N-labeled α-amanitin. A. [M+H⁺] = 919.4, [M+Na⁺] = 941.4, and [M+K⁺] = 957.4; B. [M+H⁺] = 929.4, [M+Na⁺] = 951.4, and [M+K⁺] = 967.4. (C) Overlaid spectra of ¹⁴N-amanitin and ¹⁵N-amanitin, shown in expanded scale.

Fig. 5

¹H-¹⁵N HSQC spectrum of ¹⁵N-labeled α-amanitin. The spectrum shows ten signals corresponding to the ten N atoms in α-amanitin.