Structural and Mechanistic Insights into C-S Bond Formation in Gliotoxin

Abstract

Glutathione-S-transferases (GSTs) usually detoxify xenobiotics. The human pathogenic fungus Aspergillus fumigatus however uses the exceptional GST GliG to incorporate two sulfur atoms into its virulence factor gliotoxin. Because these sulfurs are essential for biological activity, glutathionylation is a key step of gliotoxin biosynthesis. Yet, the mechanism of carbon-sulfur linkage formation from a bis-hydroxylated precursor is unresolved. Here, we report structures of GliG with glutathione (GSH) and its reaction product cyclo[-l-Phe-l-Ser]-bis-glutathione, which has been purified from a genetically modified A. fumigatus strain. The structures argue for stepwise processing of first the Phe and second the Ser moiety. Enzyme-mediated dehydration of the substrate activates GSH and a helix dipole stabilizes the resulting anion via a water molecule for the nucleophilic attack. Activity assays with mutants validate the interactions of GliG with the ligands and enrich our knowledge about enzymatic C-S bond formation in gliotoxin and epipolythiodioxopiperazine (ETP) natural compounds in general.

Keywords: Aspergillus fumigatus; carbon−sulfur bond; epipolythiodioxopiperazine; glutathione-S-transferase; mycotoxin.

Figure 1

Model for gliotoxin biosynthesis. Enzymatic coupling of the amino acids l‐Phe and l‐Ser produces the cyclic dipeptide scaffold of gliotoxin. ^[5] Upon hydroxylation of the C_α carbons of the diketopiperazine (DKP) ring by the enzyme GliC and water elimination, GliG establishes two carbon−sulfur bonds (red) per DKP with glutathione (GSH) serving as the sulfur donor. ^[6a] Referring to their site of attachment at the DKP scaffold, the two GSH molecules are termed GSH^Phe (GSH linked to the C_α atom of l‐Phe) and GSH^Ser (GSH linked to the C_α atom of l‐Ser). Stepwise decomposition of the GSH moieties and further downstream processing finally yield the disulfide‐bridged gliotoxin. ^[7d]

Figure 2

X‐ray structures of GliG with bound substrate (GSH) or reaction product (BGA). A) Ribbon illustration of GliG in complex with GSH. The two subunits of the homodimeric assembly are colored green and light blue and their N‐ as well as C‐termini are labeled. The ligand is shown as a multi‐colored balls‐and‐sticks model. Note that the active site of the light blue subunit is hidden in the back of the enzyme. B) 2 F _O−F _C electron density map for GSH and a water molecule bound to the active site of GliG (gray mesh contoured to 1 σ). Key hydrogen bonds between protein residues (shown as sticks and labeled by the one‐letter code) and the ligand are illustrated as black dashed lines. The water molecule (red sphere) is located at the positively charged N‐terminal end of helix α1. C) Ribbon illustration of GliG bound to BGA according to (A). Carbon atoms of the diketopiperazine moiety of BGA are colored purple, while those originating from GSH are depicted in pale blue. D) Connolly surface representation of the substrate binding cleft of GliG with colors indicating the electrostatic surface potential (see also Figure S5C in the Supporting Information). E) 2 F _O−F _C electron density map for BGA and a water molecule bound to GliG (gray mesh contoured to 1 σ) according to (B). The sulfur atom of the GSH^Phe moiety is located closer to the N‐terminus of helix α1 than it is the case for the GSH ligand (B). F) Numerous hydrophobic residues line the binding pocket for the Phe side chain of BGA and stabilize the ligand.

Figure 3

Activity assays and isolation of the reaction product BGA. A) Mixtures of A. fumigatus ΔgliG crude extract, GSH and purified recombinant WT or mutant GliG protein were analyzed for BGA levels by LC–HRESI‐MS to determine the relative activities of GliG variants (see method section in the Supporting Information). B) Semi‐quantitative analysis of the activity of GliG variants according to (A). The ratio of the area under the curve (AUC) of the substrate ion (m/z 267 [M+H]⁺) and the AUC of the product ion (m/z 845 [M+H]⁺) was calculated. The results are given as the mean ±standard deviation of three replicates and plotted as percent in relation to WT GliG (100 %). See also Figures S12 and S13 in the Supporting Information. C) BGA has been isolated from a genetically modified A. fumigatus strain in which the gene encoding GliK, an enzyme that acts after GliG in gliotoxin biosynthesis, has been knocked out. ^[7d] The ΔgliK strain was cultured and the accumulated BGA was extracted and purified.

Figure 4

Proposed reaction sequence for GliG. The bis‐hydroxylated precursor 1 is converted to the bis‐glutathione adduct (BGA, 2) by the action of GliG (left; solid arrows). The anticipated intermediate steps leading from 1 to 2 are shown on the right (dashed arrows). Interactions with protein side chains (labeled by the one‐letter code) are illustrated as half circles (hydrophobic contacts) or dashed lines (hydrogen bonds). Substrate 1 is bound by GliG along with GSH and upon activation by Thr23O eliminates water to yield an imine. Hereby GSH is deprotonated and the resulting sulfur anion, stabilized by the positively charged dipole of helix α1 via a water molecule, nucleophilically attacks the imine intermediate. Based on the crystallographic data, preferential glutathionylation of the Phe moiety is proposed. Once the mono‐glutathione adduct (MGA) has been formed, it is released. The final reaction product BGA is yielded by modification of a MGA molecule bound in the reverse orientation (180° rotated).