Biosynthetic gene clusters for epipolythiodioxopiperazines in filamentous fungi

Fox, Ellen M.; Howlett, Barbara J.

doi:10.1016/j.mycres.2007.08.017

Cited by 79 publications

(69 citation statements)

References 69 publications

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“…The occurrence of ETP-encoding gene clusters and studies directed towards elucidation of gliotoxin biosynthesis, and toxicity, are the focus of significant current effort [1,2,23,[29][30][31][32][33]. Thus, the development of a methodology for the specific detection of gliotoxin, especially under aqueous conditions and without the requirement for prior organic extraction and solvent removal, is significant because it enables direct identification of gliotoxin without specimen pre-treatment which may otherwise lead to gliotoxin loss or modification.…”

Section: Discussionmentioning

confidence: 99%

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

et al. 2011

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Gliotoxin is produced by non-ribosomal peptide synthesis and secreted from certain fungi, including Aspergillus fumigatus. It is an epipolythiodioxopiperazine that contains an intact disulphide bridge and is the focus of intense research as a consequence of its negative immunomodulatory properties. Gliotoxin detection is generally enabled by reversed-phase-high-performance liquid chromatography (RP-HPLC), with absorbance detection (220-280 nm), or liquid chromatography-mass spectrometry, yet detection is not readily achievable by matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-ToF MS). We have developed a single-pot derivatisation strategy which uses sodium borohydride-mediated reduction of gliotoxin followed by immediate alkylation of exposed thiols by 5′-iodoacetamidofluorescein to yield a stable product, diacetamidofluorescein-gliotoxin (GT-(AF) 2 ), of molecular mass 1103.931 Da ((M+H)+). This product is readily detectable by RP-HPLC and exhibits a 6.8-fold increase in molar absorptivity compared with gliotoxin, which results in a higher sensitivity of detection (40 ng; 125 pmoL). GT-(AF) 2 also fluoresces (excitation/emission, 492:518 nm). Unlike free gliotoxin, the product (>800 fmol) is detectable by MALDI-ToF MS. Sporidesmin A can also be detected by RP-HPLC and MALDI-ToF MS (>530 fmol) using this strategy. We also demonstrate that the strategy facilitates detection of gliotoxin (mean± SD = 3.55 ± 0.07 μg 100 μL −1 ; n = 2) produced by A. fumigatus, without the requirement for organic extraction of culture supernatants and associated solvent removal. GT-(AF) 2 is also detectable (150 ng; 460 pmol) by thinlayer chromatography.

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Section: Discussionmentioning

confidence: 99%

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

et al. 2011

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“…Putative biosynthetic gene clusters for sirodesmin and gliotoxin identified from Leptosphaeria maculans and Aspergillus fumigatus , respectively, are given in Figure 23. 108-110 Based on recent advances to our understanding of gliotoxin biosynthesis and the identification of homologs in the sirodesmin gene cluster, we are able to predict many of the experimentally unverified steps in the biosynthesis of sirodesmin PL (Scheme 3). …”

Section: Biosynthetic Investigations Of Epidithiodioxopiperazinesmentioning

confidence: 99%

Epidithiodioxopiperazines. occurrence, synthesis and biogenesis

2014

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Epidithiodioxopiperazine alkaloids possess an astonishing array of molecular architecture and generally exhibit potent biological activity. Nearly twenty distinct families have been isolated and characterized since the seminal discovery of gliotoxin in 1936. Numerous biosynthetic investigations offer a glimpse at the relative ease with which Nature is able to assemble this class of molecules, while providing synthetic chemists inspiration for the development of more efficient syntheses. Herein, we discuss the isolation and characterization, proposed fungal biogeneses, and total syntheses of epidithiodioxopiperazines.

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“…Inactivation of the non-ribosomal peptide synthase gene sirP from this cluster results in abolishment of sirodesmin production, demonstrating its involvement in the biosynthesis of sirodesmin (Gardiner et al, 2004). From this cluster, a putative prenyltransferase gene sirD has been identified, and proposed to be responsible either for the O-prenylation of L-Tyr, resulting in the formation of 4-Odimethylallyl-L-Tyr, or for the O-prenylation of the cyclic dipeptide cyclo-L-Tyr-L-Ser, resulting in the formation of phomamide (Fox & Howlett, 2008;Gardiner et al, 2004). Experimental data on the function of SirD have not been found in the literature.…”

Section: Introductionmentioning

confidence: 99%

A tyrosine O-prenyltransferase catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL

Kremer

2010

Microbiology

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A putative prenyltransferase gene sirD has been identified in the gene cluster encoding the biosynthesis of the phytotoxin sirodesmin PL in Leptosphaeria maculans. The gene product was found to comprise 449 aa, with a molecular mass of 51 kDa. In this study, the coding region of sirD was amplified by PCR from cDNA, cloned into pQE70, and overexpressed in Escherichia coli. The overproduced protein was purified to apparent homogeneity, and characterized biochemically. The dimeric recombinant SirD was found to catalyse the O-prenylation of L-Tyr in the presence of dimethylallyl diphosphate; this was demonstrated unequivocally by isolation and structural elucidation of the enzymic product. Therefore, SirD catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL. K m values for L-Tyr and dimethylallyl diphosphate were determined as 0.13 and 0.17 mM, respectively. Interestingly, SirD was found to share significant sequence similarity with indole prenyltransferases, which catalyse prenyl transfer reactions onto different positions of indole rings. In contrast to indole prenyltransferases, which accept indole derivatives, but not Tyr or structures derived thereof, as substrates, SirD also prenylated L-Trp, resulting in the formation of 7-dimethylallyltryptophan. A K m value of 0.23 mM was determined for L-Trp. Turnover numbers of 1.0 and 0.06 S "1 were calculated for L-Tyr and L-Trp, respectively.

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Biosynthetic gene clusters for epipolythiodioxopiperazines in filamentous fungi

Cited by 79 publications

References 69 publications

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

Epidithiodioxopiperazines. occurrence, synthesis and biogenesis

A tyrosine O-prenyltransferase catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL

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