32 research outputs found
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 <sup>3</sup><i>J</i><sub>6–7</sub> 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 IC<sub>50</sub> values of 441 ± 23 and 549 ± 2.5 μM, respectively.
Their activity was comparable to that of acarbose (IC<sub>50</sub> = 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<sup>-1</sup>). OD<sub>600</sub> 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, <sup>3</sup><i>J</i><sub>C,H</sub>, and <sup>3</sup><i>J</i><sub>H,H</sub> 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><sub>d</sub>) of 18.25
and 19.40 nM, respectively, 70-fold higher than that of chlorpromazine
(<i>K</i><sub>d</sub> = 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 <sup>1</sup>H–<sup>1</sup>H NMR coupling constant analyses, which reinforce the stereochemical
assignment. The potential affinity of <b>1</b> and <b>2</b> with (Ca<sup>2+</sup>)<sub>4</sub>-<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><sub>d</sub>) of 0.25 ± 0.04 μM, close to that of chlorpromazine
(<i>K</i><sub>d</sub> = 0.64 ± 0.03 μM), a classical
CaM inhibitor. The stoichiometry ratio of <b>1</b> to (Ca<sup>2+</sup>)<sub>4</sub>-<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<sup>-1</sup>). 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