Structural, mechanistic and functional insight into gliotoxin bis-thiomethylation in Aspergillus fumigatus.

Stephen K Dolan,Rebecca A Owens,Wulf Blankenfeldt,Vanessa Hering,Sean Doyle,Tobias Bock,Gary W Jones

doi:10.1098/rsob.160292

Abstract

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus. Self-resistance against gliotoxin is effected by the gliotoxin oxidase GliT, and attenuation of gliotoxin biosynthesis is catalysed by gliotoxin S-methyltransferase GtmA. Here we describe the X-ray crystal structures of GtmA-apo (1.66 Å), GtmA complexed to S-adenosylhomocysteine (1.33 Å) and GtmA complexed to S-adenosylmethionine (2.28 Å), providing mechanistic insights into this important biotransformation. We further reveal that simultaneous elimination of the ability of A. fumigatus to dissipate highly reactive dithiol gliotoxin, via deletion of GliT and GtmA, results in the most significant hypersensitivity to exogenous gliotoxin observed to date. Indeed, quantitative proteomic analysis of ΔgliT::ΔgtmA reveals an uncontrolled over-activation of the gli-cluster upon gliotoxin exposure. The data presented herein reveal, for the first time, the extreme risk associated with intracellular dithiol gliotoxin biosynthesis—in the absence of an efficient dismutation capacity. Significantly, a previously concealed protective role for GtmA and functionality of ETP bis-thiomethylation as an ancestral protection strategy against dithiol compounds is now evident.

Highlights

Ascomycetes constitute the largest phylum of the fungal kingdom and produce a copious array of natural products
We further reveal that simultaneous elimination of the ability of A. fumigatus to dissipate highly reactive dithiol gliotoxin, via deletion of GliT and GtmA, results in the most significant hypersensitivity to exogenous gliotoxin observed to date
We subsequently demonstrated that GtmA-mediated bis-thiomethylation of gliotoxin by A. fumigatus regulates the production of this toxin in A. fumigatus by disrupting a positive feedback loop which normally potentiates gliotoxin biosynthesis [12]

Summary

Introduction

Ascomycetes constitute the largest phylum of the fungal kingdom and produce a copious array of natural products. Many of these compounds are known as clinically important drugs or industrial chemicals, several natural products are potent toxins that pose substantial threats to human food supplies and health [1]. Production of these natural products requires a carefully orchestrated system to balance biosynthesis while avoiding self-harm from endogenous accumulation of toxic natural product precursors [2]. The stereochemically complex core of ETPs, coupled with their potent biological activities, make these compounds an attractive target for drug leads [7]

Methods

Results

Conclusion