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Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis.

Gemmecker S, Schaub P, Koschmieder J, Brausemann A, Drepper F, Rodriguez-Franco M, Ghisla S, Warscheid B, Einsle O, Beyer P - PLoS ONE (2015)

Bottom Line: Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase.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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Cell Biology, University of Freiburg, Freiburg, Germany.

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.

No MeSH data available.


Preliminary structural analysis of PDS-His6 by X-ray crystallography.A, Stereo representation of a representative section of the experimental electron density map as a blue mesh, with initial α-helical models shown as cylinders. B, Superposition of a preliminary PDS-model shown as yellow cylinders with the structure of CRTI (PDB-ID: 4DGK) in blue, displaying the high similarity in the overall structure of the two enzymes. C; Cartoon representation of a superposition of CRTI in blue with an oxidoreductase from Methanosarcina mazei (PDB-ID: 3KA7) in grey and its FAD cofactor in yellow.
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pone.0131717.g007: Preliminary structural analysis of PDS-His6 by X-ray crystallography.A, Stereo representation of a representative section of the experimental electron density map as a blue mesh, with initial α-helical models shown as cylinders. B, Superposition of a preliminary PDS-model shown as yellow cylinders with the structure of CRTI (PDB-ID: 4DGK) in blue, displaying the high similarity in the overall structure of the two enzymes. C; Cartoon representation of a superposition of CRTI in blue with an oxidoreductase from Methanosarcina mazei (PDB-ID: 3KA7) in grey and its FAD cofactor in yellow.

Mentions: Diffracting single yellow colored crystals of PDS-His6 were obtained by sitting-drop vapor diffusion, and the mercuric compound thiomersal was successfully employed to address the crystallographic phase problem. In the best diffraction data sets available at present, the anomalous signal of mercury extended to a maximum resolution of 7.5 Å, and phase information calculated on this basis was iteratively extended to yield an experimental electron density map at 6 Å resolution (Fig 7A). Data collection statistics are summarized in Table 2. While this was not yet of sufficient quality to build an atomic model for the enzyme, the map showed unambiguous solvent boundaries and continuous stretches of electron density that likely represent α-helices. This led to a first low-resolution model for PDS-His6 with a kinked two-domain architecture that underlines a homotetrameric arrangement of the protein in the crystals (Fig 7A). The homotetramer is generated through a crystallographic two-fold axis. Even at the present resolution, the model obtained for PDS-His6 shows clear similarities to the structure of CRTI (Fig 7B).


Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis.

Gemmecker S, Schaub P, Koschmieder J, Brausemann A, Drepper F, Rodriguez-Franco M, Ghisla S, Warscheid B, Einsle O, Beyer P - PLoS ONE (2015)

Preliminary structural analysis of PDS-His6 by X-ray crystallography.A, Stereo representation of a representative section of the experimental electron density map as a blue mesh, with initial α-helical models shown as cylinders. B, Superposition of a preliminary PDS-model shown as yellow cylinders with the structure of CRTI (PDB-ID: 4DGK) in blue, displaying the high similarity in the overall structure of the two enzymes. C; Cartoon representation of a superposition of CRTI in blue with an oxidoreductase from Methanosarcina mazei (PDB-ID: 3KA7) in grey and its FAD cofactor in yellow.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492965&req=5

pone.0131717.g007: Preliminary structural analysis of PDS-His6 by X-ray crystallography.A, Stereo representation of a representative section of the experimental electron density map as a blue mesh, with initial α-helical models shown as cylinders. B, Superposition of a preliminary PDS-model shown as yellow cylinders with the structure of CRTI (PDB-ID: 4DGK) in blue, displaying the high similarity in the overall structure of the two enzymes. C; Cartoon representation of a superposition of CRTI in blue with an oxidoreductase from Methanosarcina mazei (PDB-ID: 3KA7) in grey and its FAD cofactor in yellow.
Mentions: Diffracting single yellow colored crystals of PDS-His6 were obtained by sitting-drop vapor diffusion, and the mercuric compound thiomersal was successfully employed to address the crystallographic phase problem. In the best diffraction data sets available at present, the anomalous signal of mercury extended to a maximum resolution of 7.5 Å, and phase information calculated on this basis was iteratively extended to yield an experimental electron density map at 6 Å resolution (Fig 7A). Data collection statistics are summarized in Table 2. While this was not yet of sufficient quality to build an atomic model for the enzyme, the map showed unambiguous solvent boundaries and continuous stretches of electron density that likely represent α-helices. This led to a first low-resolution model for PDS-His6 with a kinked two-domain architecture that underlines a homotetrameric arrangement of the protein in the crystals (Fig 7A). The homotetramer is generated through a crystallographic two-fold axis. Even at the present resolution, the model obtained for PDS-His6 shows clear similarities to the structure of CRTI (Fig 7B).

Bottom Line: Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase.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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Cell Biology, University of Freiburg, Freiburg, Germany.

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.

No MeSH data available.