α‑Glucosidase Inhibitors from a <i>Xylaria feejeensis</i> Associated with <i>Hintonia latiflora</i>

Two new compounds, pestalotin 4′-<i>O</i>-methyl-β-mannopyranoside (<b>1</b>) and 3<i>S</i>,4<i>R</i>-(+)-4-hydroxymellein (<b>2</b>), were isolated from an organic extract of a <i>Xylaria feejeensis</i>, which was isolated as an endophytic fungus from <i>Hintonia latiflora.</i> In addition, the known compounds 3<i>S</i>,4<i>S</i>-(+)-4-hydroxymellein (<b>3</b>), 3<i>S</i>-(+)-8-methoxymellein (<b>4</b>), and the quinone derivatives 2-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (<b>5</b>), 4<i>S</i>,5<i>S</i>,6<i>S</i>-4-hydroxy-3-methoxy-5-methyl-5,6-epoxycyclohex-2-en-1-one (<b>6</b>), and 4<i>R</i>,5<i>R</i>-dihydroxy-3-methoxy-5-methylcyclohexen-2-en-1-one (<b>7</b>) were obtained. The structures of <b>1</b> and <b>2</b> were elucidated using a set of spectroscopic and spectrometric techniques. The absolute configuration of the stereogenic centers of <b>1</b> and <b>2</b> was determined using ECD spectroscopy combined with time-dependent density functional theory calculations. In the case of <b>1</b>, comparison of the experimental and theoretical ³<i>J</i>_6–7 coupling constants provided further evidence for the stereochemical assignments. Compounds <b>2</b> and <b>3</b> inhibited <i>Saccharomyces cerevisiae </i>α-glucosidase (αGHY), with IC₅₀ values of 441 ± 23 and 549 ± 2.5 μM, respectively. Their activity was comparable to that of acarbose (IC₅₀ = 545 ± 19 μM), used as positive control. Molecular docking predicted that both compounds bind to αGHY in a site different from the catalytic domain, which could imply an allosteric type of inhibition

Two Horizontally Transferred Xenobiotic Resistance Gene Clusters Associated with Detoxification of Benzoxazolinones by <i>Fusarium</i> Species

<div><p>Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that <i>Fusarium verticillioides</i>, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, <i>FDB1</i> and <i>FDB2</i>, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of <i>F</i>. <i>verticillioides</i> exposed to BOA identified the induction of multiple genes at <i>FDB1</i> and <i>FDB2</i>, indicating the loci consist of gene clusters. One of the <i>FDB1</i> cluster genes encoded a protein having domain homology to the metallo-β-lactamase (MBL) superfamily. Deletion of this gene (<i>MBL1</i>) rendered <i>F</i>. <i>verticillioides</i> incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other <i>FDB1</i> cluster genes, in particular <i>AMD1</i> and <i>DLH1</i>, did not affect BOA degradation. Phylogenetic analyses and topology testing of the <i>FDB1</i> and <i>FDB2</i> cluster genes suggested two horizontal transfer events among fungi, one being transfer of <i>FDB1</i> from <i>Fusarium</i> to <i>Colletotrichum</i>, and the second being transfer of the <i>FDB2</i> cluster from <i>Fusarium</i> to <i>Aspergillus</i>. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or virulence.</p></div

Growth curve analysis of deletion strains in comparison to wild type.

<p>Strains were monitored for 100 h in PDB amended with BOA (0.5 mg ml^-1). OD₆₀₀ measurements recorded every 2 h were plotted (mean ± standard deviation). For this experiment BOA was dissolved in DMSO, and a DMSO-only control for wild-type strain FRC M-3125 is shown (grey line). FRC M-3125 in PDB with BOA is shown with a black line. The <i>Δamd1</i> strain (Δ08289–2) is the orange line with triangles. The <i>Δdlh1</i> mutant (Δ08290–11) is the blue line with squares. The <i>Δmbl1</i> mutants, Δ08291–33 and Δ08291–36, are the brown line with circle markers and red line with × markers, respectively. The <i>Δmbl1</i>::<i>MBL1</i> complemented strain (Δ08291–36::C2) is the green line with × markers. Experiment was conducted twice with similar results. Data from one experiment is presented.</p

Absolute Configuration of Acremoxanthone C, a Potent Calmodulin Inhibitor from <i>Purpureocillium lilacinum</i>

Bioassay-guided fractionation of an extract prepared from the culture medium and mycelium of <i>Purpureocillium lilacinum</i> allowed the isolation of two calmodulin (CaM) inhibitors, namely, acremoxanthone C (<b>1</b>) and acremonidin A (<b>2</b>). The absolute configuration of <b>1</b> was established as 2<i>R</i>, 3<i>R</i>, 1′<i>S</i>, 11′<i>S</i>, and 14′<i>R</i> through extensive NMR spectroscopy and molecular modeling calculations at the DFT B3LYP/DGDZVP level, which included the comparison between theoretical and experimental specific rotation, ³<i>J</i>_C,H, and ³<i>J</i>_H,H values. Compounds <b>1</b> and <b>2</b> bind to the human calmodulin (<i>h</i>CaM) biosensor <i>h</i>CaM M124C-<i>mBBr</i>, with dissociation constants (<i>K</i>_d) of 18.25 and 19.40 nM, respectively, 70-fold higher than that of chlorpromazine (<i>K</i>_d = 1.24 μM), used as positive control. Docking analysis using AutoDock 4.2 predicted that <b>1</b> and <b>2</b> bind to CaM at a similar site to that which KAR-2 binds, which is unusual. Furthermore, a novel, sensible, and specific fluorescent biosensor of <i>h</i>CaM, i<i>.</i>e<i>., h</i>CaM T110C-<i>mBBr</i>, was constructed; this device is labeled at a site where classical inhibitors do not interact and was successfully applied to measure the interaction of <b>1</b> with CaM. This is the first report of xanthone–anthraquinone heterodimers in species of <i>Paecilomyces</i> or <i>Purpureocillium</i> genera

(+)-Ascosalitoxin and Vermelhotin, a Calmodulin Inhibitor, from an Endophytic Fungus Isolated from <i>Hintonia latiflora</i>

Chemical investigation of the endophytic MEXU 26343, isolated from the medicinal plant <i>Hintonia latiflora</i>, yielded the known polyketide vermelhotin (<b>1</b>) and a new salicylic aldehyde derivative, namely, 9<i>S</i>,11<i>R</i>-(+)-ascosalitoxin (<b>2</b>). The structure and absolute configuration of the new compound were established through extensive NMR spectroscopy and molecular modeling calculations at the DFT B3LYP/DGDZVP level, which included the comparison between theoretical and experimental optical rotation values. In addition, chemical transformations of <b>2</b> yielded suitable derivatives for NOESY and ¹H–¹H NMR coupling constant analyses, which reinforce the stereochemical assignment. The potential affinity of <b>1</b> and <b>2</b> with (Ca²⁺)₄-<i>h</i>CaM in solution was measured using the fluorescent biosensor <i>h</i>CaM M124C-<i>mBBr</i>. The results showed that <b>1</b> bound to the protein with a dissociation constant (<i>K</i>_d) of 0.25 ± 0.04 μM, close to that of chlorpromazine (<i>K</i>_d = 0.64 ± 0.03 μM), a classical CaM inhibitor. The stoichiometry ratio of <b>1</b> to (Ca²⁺)₄-<i>h</i>CaM was 1:4, similar to other well-known CaM ligands

Protein based phylogeny of FVEG_08290 (<i>DLH1</i>) and FVEG_12625 (<i>DLH2</i>).

<p>The PhyML cladogram is shown with bootstrap values (200 replications) indicated for branches having ≥80% support. Bayesian posterior probabilities are also indicated for those branches. <i>Zymoseptoria tritici</i> XP_003850758 was the designated outgroup.</p

Complementation assay of the <i>F</i>. <i>verticillioides fdb1</i> mutant with clones derived from transposon mutagenesis of cosmid F5D9.

<p>(A) Chromosome 10 genes FVEG_08287 –FVEG_08295 (black block arrows) from the <i>FDB1</i> gene cluster are shown in their transcriptional orientations. Cosmid clone F5D9 (long, thin black arrow) includes full-length copies of these genes except for FVEG_08287, which is truncated. Green and red triangles indicate the positions of transposon insertions in 20 different F5D9-derived clones following mutagenesis. Green indicates transposon insertions that did not affect complementation of the <i>fdb1</i> mutant, whereas red indicates insertions that affected complementation. (B) Growth phenotypes of strain AEG 74-A4-3 transformed separately with clone F5D9 and 20 F5D9-derived clones on BOA medium (0.9 mg ml^-1). Transformation of strain AEG 74-A4-3 with clone F5D9 and most F5D9-derived clones (green triangles) restored wild-type growth in the presence of BOA. Four F5D9-derived clones (red triangles; insertions 244, 264, 260 and 375) did not restore wild-type growth in the presence of BOA. The four latter transformants were also not able to metabolize BOA.</p

Protein based phylogeny of FVEG_08291 (<i>MBL1</i>) and FVEG_12637 (<i>MBL2</i>).

<p>The PhyML cladogram is shown with bootstrap values (200 replications) indicated for branches having ≥80% support. Bayesian posterior probabilities are also indicated for those branches. <i>Zymoseptoria tritici</i> XP_003854792 was the designated outgroup.</p

HPLC analysis of BOA detoxification into HPMA by deletion mutants.

<p>Chromatograms of (A) BOA and (B) HPMA standards (retention times of 10.9 and 6.69 min, respectively) in comparison to PDB amended with BOA that was either (C) left uninoculated (PDB+BOA) or was inoculated with (D) wild-type FRC M-3125 or the deletion mutants of (E) FVEG_08289 (<i>AMD1</i>), (F) FVEG_08290 (<i>DLH1</i>), or (G-H) FVEG_08291 (<i>MBL1</i>; two different deletion mutants). Wild type, <i>Δamd1</i>, and <i>Δdlh1</i> were all able to fully metabolize BOA to HPMA. Two <i>Δmbl1</i> mutants, Δ08291–33 and Δ08291–36, were unable to metabolize BOA. (I) Add-back of <i>MBL1</i> (Δ08291–36::C2) complemented the deletion.</p

Gene expression analysis of <i>F</i>. <i>verticillioides</i> exposed to BOA.

<p>Microarray expression values for selected genes of <i>F</i>. <i>verticillioides</i> grown for two hours in PDB supplemented with either ethanol alone (blue) or BOA dissolved in ethanol (red). Gene expression values are shown for the proposed <i>FDB1</i> cluster (FVEG_08287 to FVEG_08295) and <i>FDB2</i> cluster (FVEG_12625 to FVEG_12641) as well as for genes flanking each end of the clusters. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147486#pone.0147486.s010" target="_blank">S1 Table</a> for more details on the genes and their fold change in expression.</p