Reconstitution of the early steps of gliotoxin biosynthesis in Aspergillus nidulans reveals the role of the monooxygenase GliC

Abstract

The gliotoxin, a member of the epipolythiodioxopiperazine (ETP), has received considerable attention from the scientific community for its wide range of biological activity. Despite the identification of gliotoxin cluster, however, the sequence of steps in the gliotoxin biosynthesis has remained elusive. As an alternative to the gene knock out and biochemical approaches used so far, here we report using a heterologous expression approach to determine the sequence of the early steps of gliotoxin biosynthesis in A. nidulans. We identified the GliC, a monooxygenases that involved in the second step of gliotoxin biosynthesis pathway through the catalyzing the hydroxylation at the α-position of l-Phe.

Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus

“…In effect, the A. fumigatus ⌬gliK strain is a "GliT-lite" mutant. Moreover, this observation supports the hypothesis of the requirement for de novo gliotoxin biosynthesis to effect maximum gliotoxin-mediated growth inhibition, as significantly less GliT is required for GT-(SH) 2 (Fig. 5D) (5).…”

“…Proteins found to be significantly differentially expressed (P Ͻ 0.05) after analysis by using Progenesis SameSpot software are numbered. (C) In vitro GtmA activity, as determined by BmGT formation in the presence of SAM and GT-(SH) 2 , is evident in mycelial lysates from A. fumigatus ⌬gliK cells exposed to gliotoxin (5 g/ml for 3 h). Assay conditions were described previously (10).…”

“…This apparently puzzling result means that the growth-inhibitory phenotype observed upon gliotoxin exposure requires both a functional biosynthetic system and an inactive resistance system. Otherwise, if gliZ expression is intact, exogenous gliotoxin induces gli cluster expression (13,16,17) and leads to the de novo production of GT-(SH) 2 and thus an exacerbation of the growth-inhibitory effect of exogenous gliotoxin. This observation also explains why the A. fumigatus ⌬gliT strain grows a A change in abundance was considered significant at a P value of Ͻ0.05.…”

“…iosynthesis, self-protection mechanisms, and functionality of gliotoxin and related epidithiodiketopiperazine (ETP) molecular species, such as chaetocin and acetylaranotin, are attracting ever-increasing attention as a consequence of findings from highthroughput genome sequencing projects, application of gene deletion technologies, and mass spectrometric analytical methodologies (1)(2)(3)(4)(5). Indeed, existing paradigms of gliotoxin ( Fig.…”

“…1) has not been reported in most studies relating to gliT, gliA, gliK, or gliG (a glutathione S-transferase responsible for biosynthetic intermediate sulfurization via bis-glutathionylation) gene cluster deletion (4,5,7,13,15,18,19), this metabolite is readily detectable in culture supernatants of A. fumigatus (8)(9)(10). Moreover, endogenous dithiol gliotoxin [GT-(SH) 2 ] and exogenous gliotoxin can be converted to BmGT via a novel, S-adenosylmethionine (SAM)-dependent gliotoxin bis-thiomethyltransferase (GtmA) (10). Contrary to speculation in the literature (14) that deficiency of such an enzyme would induce gliotoxin sensitivity in A. fumigatus, as per deletion of gliT, no such phenotype has been observed.…”