A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore.

The model white-rot basidiomycete, Ceriporiopsis (Gelatoporia) subvermispora B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT1C1T2C2T3C3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in Aspergillus niger as a soluble and active protein. N 5-acetyl-N 5-hydroxy-l-ornithine (l-AHO) and N 5-cis-anhydromevalonyl-N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as the substrates, based on results of an in vitro substrate-dependent [32P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo-CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as the product in vitro, which was verified by liquid chromatography-high-resolution electrospray ionization-mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes.IMPORTANCE The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the Aspergillus niger/SM-Xpress-based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the >250-kDa range in soluble and active form.

Opposed effects of enzymatic gliotoxin N- and S-methylations.

Gliotoxin (1), a virulence factor of the human pathogenic fungus Aspergillus fumigatus, is the prototype of epipoly(thiodioxopiperazine) (ETP) toxins. Here we report the discovery and functional analysis of two methyl transferases (MTs) that play crucial roles for ETP toxicity. Genome comparisons, knockouts, and in vitro enzyme studies identified a new S-adenosyl-l-methionine-dependent S-MT (TmtA) that is, surprisingly, encoded outside the gli gene cluster. We found that TmtA irreversibly inactivates ETP by S-alkylation and that this detoxification strategy appears to be not only limited to ETP producers. Furthermore, we unveiled that GliN functions as a freestanding amide N-MT. GliN-mediated amide methylation confers stability to ETP, damping the spontaneous formation of tri- and tetrasulfides. In addition, enzymatic N-alkylation constitutes the last step in gliotoxin biosynthesis and is a prerequisite for the cytotoxicity of the molecule. Thus, these specialized alkylating enzymes have dramatic and fully opposed effects: complete activation or inactivation of the toxin.

Flavoenzyme-catalyzed formation of disulfide bonds in natural products.

Nature provides a rich source of compounds with diverse chemical structures and biological activities, among them, sulfur-containing metabolites from bacteria and fungi. Some of these compounds bear a disulfide moiety that is indispensable for their bioactivity. Specialized oxidoreductases such as GliT, HlmI, and DepH catalyze the formation of this disulfide bridge in the virulence factor gliotoxin, the antibiotic holomycin, and the anticancer drug romidepsin, respectively. We have examined all three enzymes by X-ray crystallography and activity assays. Despite their differently sized substrate binding clefts and hence, their diverse substrate preferences, a unifying reaction mechanism is proposed based on the obtained crystal structures and further supported by mutagenesis experiments.

A dedicated glutathione S-transferase mediates carbon-sulfur bond formation in gliotoxin biosynthesis.

Gliotoxin is a virulence factor of the human pathogen Aspergillus fumigatus , the leading cause of invasive aspergillosis. Its toxicity is mediated by the unusual transannular disulfide bridge of the epidithiodiketopiperazine (ETP) scaffold. Here we disclose the critical role of a specialized glutathione S-transferase (GST), GliG, in enzymatic sulfurization. Furthermore, we show that bishydroxylation of the diketopiperazine by the oxygenase GliC is a prerequisite for glutathione adduct formation. This is the first report of the involvement of a GST in enzymatic C-S bond formation in microbial secondary metabolism.

Conformational studies on the Delta8(E,Z)-sphingolipid desaturase from Helianthus annuus with chiral fluoropalmitic acids as mechanistic probes.

The Delta(8)-sphingolipid desaturase from sunflower (Helianthus annuus) converts phytosphinganine into a mixture of Delta(8)-(E)- and -(Z)-phytosphingenines by removal of two syn-hydrogen atoms from anti-, and gauche-conformations of the substrate. With chiral (R)-6-, (S)-6-, (R)-7-, and (S)-7-fluoropalmitic acids the importance of conformations for the formation of (E)- and (Z)-isomers was investigated by using growing yeast cells expressing the desaturase from H. annuus. The fluoropalmitic acids were readily incorporated into a series of fluorinated phytosphinganines. The desaturation products of the major C(18)-fluorophytosphinganine demonstrate that different conformations of the relevant aliphatic segment of the sphingolipids can be exposed to the active center of the enzyme resulting in (E)- or (Z)-fluoroalkenes. The presence of a fluorine atom at the position of the initial hydrogen removal C8-H(R) led to a complete suppression of the desaturation reaction, while replacement of C8-H(S) with fluorine generated a mixture of mainly (Z)- and trace amounts of (E)-fluoroolefine. Fluorine at C9 of the phytosphinganine precursors did not interfere with the initial C-H activation step and produced (E)- and (Z)-fluoroalkenes in the same ratio as observed for the nonfluorinated precursors. Hydroxylated byproducts of the desaturation process were not observed. These results strongly support the importance of conformations of the transition states during desaturation as the relevant criterion for the relative ratio of (E)- and (Z)-alkenes.

1-phenylethyl isocyanate is a powerful reagent for the chiral analysis of secondary alcohols and hydroxy fatty acids with remote stereogenic centres.

1-phenylethyl isocyanate (1-PEIC), a chiral derivatisation reagent for the resolution of secondary alcohols is a powerful tool to determine the configuration and enantiomeric excess of medium- to long-chain secondary alcohols by capillary gas chromatography. The separation of 1-phenylethylcarbamates (1-PECs) of secondary alcohols was systematically evaluated depending on the position of the stereogenic centre in the molecule, namely in alkanols (C(15)-C(18)), alkenols (C(15)-C(18)) and hydroxy fatty acids (C(14)-C(18)). The successful separation of the diastereomeric carbamates of (+/-)-heptadecan-7-ol or (+/-)-12-hydroxyoctadecanoic acid methyl ester by gas liquid chromatography demonstrates the unique separation power for 1-PECs for analytes with remote stereogenic centres. Saturated derivatives showed consistently higher resolution factors than the corresponding unsaturated derivatives.

Draft Genome Sequence and Gene Annotation of the Entomopathogenic Fungus Verticillium hemipterigenum.

Verticillium hemipterigenum (anamorph Torrubiella hemipterigena) is an entomopathogenic fungus and produces a broad range of secondary metabolites. Here, we present the draft genome sequence of the fungus, including gene structure and functional annotation. Genes were predicted incorporating RNA-Seq data and functionally annotated to provide the basis for further genome studies.

Efficient and flexible synthesis of chiral gamma- and delta-lactones.

An efficient and highly flexible synthesis for chiral gamma- and delta-lactones with high enantiomeric purity is described (>99% ee and 57-87% overall yield). The protocol involves alkylation of chiral 1,2-oxiranes with terminally unsaturated Grignard reagents. Subsequent oxidative degradation (OsO(4)-Oxone) of the terminal double bond from chiral alk-1-en-5-ols and alk-1-en-6-ols affords 4- or 5-hydroxy acids and gamma- and delta-lactones after acidic workup. The flexibility and efficiency of the protocol is illustrated by the synthesis of several alkanolides and alkenolides, hydroxy fatty acids and dihydroisocoumarins.

Evidence for a radical mechanism of the dechlorination of chlorinated propenes mediated by the tetrachloroethene reductive dehalogenase of Sulfurospirillum muftivorans.

The reductive dehalogenation of chlorinated propenes was studied with the tetrachloroethene reductive dehalogenase purified from Sulfurospirillum multivorans to obtain indications for a radical mechanism of this reaction. When reduced methyl viologen (MV), which is a radical cation, was applied as electron donor for the reduction of different chloropropenes, a significant part of MV could not be rereduced with Ti(III) citrate, indicating that a part of the MV was consumed in a side reaction. Mass spectrometric analysis of assays with MV as electron donor revealed the formation of side products, the masses of which might account for the formation of adducts from a chloropropenyl radical and reduced methyl viologen. With Ti(III) citrate as sole electron donor, 2,3-dichloropropene was reduced and as a side product, 2,5-dichloro-1,5-hexadiene was formed demonstrating that the reductive dechlorination of 2,3-dichloropropene proceeds via a radical reaction mechanism. The results support different dehalogenation mechanisms forthe reductive dechlorination of chloropropenes and halogenated ethenes.