Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FAD(red), while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.
%0 Journal Article
%1 gemmeckerPhytoeneDesaturaseOryza2015
%A Gemmecker, Sandra
%A Schaub, Patrick
%A Koschmieder, Julian
%A Brausemann, Anton
%A Drepper, Friedel
%A Rodriguez-Franco, Marta
%A Ghisla, Sandro
%A Warscheid, Bettina
%A Einsle, Oliver
%A Beyer, Peter
%C United States
%D 2015
%J PloS one
%K & Binding,Protein Biopolymers/*chemistry,Cell Conformation,to_read Electron Electrophoresis,Oryza/*enzymology,Oxidoreductases/*chemistry/isolation Gel Membrane/enzymology,Crystallography Polyacrylamide Scanning,Native Spectrometry,Microscopy X-Ray,Mass purification/metabolism,Protein
%N 7
%P e0131717
%R 10.1371/journal.pone.0131717
%T Phytoene Desaturase from Oryza Sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis.
%V 10
%X Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FAD(red), while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.
@article{gemmeckerPhytoeneDesaturaseOryza2015,
abstract = {Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FAD(red), while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.},
added-at = {2024-05-17T13:01:35.000+0200},
address = {United States},
author = {Gemmecker, Sandra and Schaub, Patrick and Koschmieder, Julian and Brausemann, Anton and Drepper, Friedel and {Rodriguez-Franco}, Marta and Ghisla, Sandro and Warscheid, Bettina and Einsle, Oliver and Beyer, Peter},
biburl = {https://www.bibsonomy.org/bibtex/253f9bb4bc52bfdb1880e1f788620cebe/warscheidlab},
doi = {10.1371/journal.pone.0131717},
interhash = {b33a99273fdb57a04adf7b1abca7adf8},
intrahash = {53f9bb4bc52bfdb1880e1f788620cebe},
issn = {1932-6203},
journal = {PloS one},
keywords = {& Binding,Protein Biopolymers/*chemistry,Cell Conformation,to_read Electron Electrophoresis,Oryza/*enzymology,Oxidoreductases/*chemistry/isolation Gel Membrane/enzymology,Crystallography Polyacrylamide Scanning,Native Spectrometry,Microscopy X-Ray,Mass purification/metabolism,Protein},
langid = {english},
number = 7,
pages = {e0131717},
pmcid = {PMC4492965},
pmid = {26147209},
timestamp = {2024-05-17T13:01:35.000+0200},
title = {Phytoene {{Desaturase}} from {{Oryza}} Sativa: {{Oligomeric Assembly}}, {{Membrane Association}} and {{Preliminary 3D-Analysis}}.},
volume = 10,
year = 2015
}