22 research outputs found
A Short Access to the Skeleton of Elisabethin A and Formal Syntheses of Elisapterosin B and Colombiasin A
A short
stereoselective synthesis of the Elisabethin A skeleton <b>4</b> is described, which opens a formal access to the diterpenes Elisapterosin
B and Colombiasin A as well. Key reactions were an intermolecular <i>endo</i>-selective Diels–Alder reaction to generate the
decalin part of the molecule, a chemo- and diastereoselective allylation
of an aldehyde with allylzinc, a palladium ene annulation of the cyclopentane
ring, and a novel sulfonium ylide induced fragmentation of a polycyclic
ketone. Additional insights have been gained for the crucial epimerization
at C-2
Oxidation of methionines.
<p>Methionine-containing peptide fragments detected that show a mass increase of 16 or 32 Da after proteolytic digestion of <i>Tm</i>POx (inactivated during turnover of 100 mM D-glucose, treated with endogenous H<sub>2</sub>O<sub>2</sub> or unaffected). Proteolytic digestion was performed with trypsin or Asp-N protease as indicated. The last column indicates whether the particular Met is considerably oxidised during substrate turnover and by H<sub>2</sub>O<sub>2</sub> treatment compared to the unaffected sample (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.g002" target="_blank">Fig 2</a>).</p
Identification of the exact site of oxidation in the peptide <sup>452</sup>DAFSYGAVQQSIDSR<sup>466</sup> by LC-ESI-MS/MS.
<p>The nearly complete series of C-terminal y-ions in the MS/MS spectrum of the double-charged ion of 830.4 confirms the peptide sequence. The +16 Da mass shift found for the ion y<sub>13</sub> further indicates that Phe454 is oxidised during substrate turnover and by H<sub>2</sub>O<sub>2</sub> treatment.</p
Active-site geometry of pyranose oxidase from <i>T</i>. <i>multicolor</i>.
<p>In the closed form of the active-site loop of pyranose oxidase from <i>T</i>. <i>multicolor</i> (<i>Tm</i>POx, PDB code 1TT0; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>]), which is thought to be relevant for the oxidative half-reaction of POx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>], Phe454 is positioned in the direct vicinity of the isoalloxazine ring and the C4a/N5 locus, at which oxygen is reduced. The figure was generated using PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>).</p
Accessibility of methionine residues.
<p>Surface of the <i>T</i>. <i>multicolor</i> POx monomer in the vicinity of (<b>A</b>) Met417, showing the surface-exposed sulphur atom which is oxidised by H<sub>2</sub>O<sub>2</sub>, and (<b>B</b>) Met380 with its sulphur-containing side chain pointing towards the interior of the polypeptide matrix, in which it is buried and hence is not accessible from the surface.</p
Mass spectrometric identification of methionine residues oxidized by H<sub>2</sub>O<sub>2</sub> during <i>Tm</i>POx inactivation.
<p>MALDI MS spectra were measured for unaffected POx (<b>A</b>), for POx inactivated during D-glucose oxidation (<b>B</b>) or for POx inactivated by endogenous H<sub>2</sub>O<sub>2</sub> (<b>C</b>). The selected MALDI spectra in the left panel illustrate that Met497 of the tryptic peptide ITDAYNMPQPTFDFR with a theoretical MH<sup>+</sup> of 1815.8 was extensively oxidised in <i>Tm</i>POx inactivated either during substrate turnover (<b>B</b>, left panel) or by H<sub>2</sub>O<sub>2</sub> treatment (<b>C</b>, left panel). In contrast, some methionine residues were found not to be oxidised during <i>Tm</i>POx inactivation as shown for Met74 of the peptide VAMFDIGEIDSGLK having a MH<sup>+</sup> of 1494.8 (right panel). The small signals at m/z 1510.8 are related to the oxidized form of the peptide generated due to the presence of air oxygen.</p
3D Structure of GalOx of <i>F. oxysporum</i>.
<p>A: Overall structure showing the predominantly β-structure. The N-terminus, C-terminus and the copper atom in the active site are highlighted. B: The active site of GalOx showing the copper ligands and the thioether cross-link. The structural model was generated by homology modelling based on the published structure of mature GalOx from <i>F. graminearum</i> (PDB 1gog) using SWISS_MODEL.</p
Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i>
<p>Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i></p
Purification of recombinant GalOx expressed in <i>E. coli.</i>
<p>Purification of recombinant GalOx expressed in <i>E. coli.</i></p
Apparent kinetic constants of GalOx produced in <i>E. coli</i> for several electron donor substrates.
<p>Apparent kinetic constants of GalOx produced in <i>E. coli</i> for several electron donor substrates.</p