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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.


Related in: MedlinePlus

Electron microscopy of PDS-His6.A, negative staining. Examples of rings (white arrows) and stacks of rings (black arrows) are indicated. The inset shows examples of stacks (upper row) and rings (lower two rows) at higher magnification; each picture represents an area of 20 x 20 nm. The bar refers to the overview and represents 100 nm. B, Freeze-fracture scanning EM. Left, membrane fracture faces of liposomes containing bound PDS-His6 showing the absence of transmembrane particles. Right, membrane surfaces exposed after sublimation. The arrow points to the surface/fracture face boundary. Particles of homogenous size are seen on the surface. Bar represents 200 nm.
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pone.0131717.g005: Electron microscopy of PDS-His6.A, negative staining. Examples of rings (white arrows) and stacks of rings (black arrows) are indicated. The inset shows examples of stacks (upper row) and rings (lower two rows) at higher magnification; each picture represents an area of 20 x 20 nm. The bar refers to the overview and represents 100 nm. B, Freeze-fracture scanning EM. Left, membrane fracture faces of liposomes containing bound PDS-His6 showing the absence of transmembrane particles. Right, membrane surfaces exposed after sublimation. The arrow points to the surface/fracture face boundary. Particles of homogenous size are seen on the surface. Bar represents 200 nm.

Mentions: Negative staining of the higher-order oligomeric species purified in the presence of norflurazon revealed a distribution of particles, among which two recurrent patterns were observed consisting of rings (white arrows) and stacks (black arrows; Fig 5A). Rings appeared mostly four- membered, with a diameter of 11.8 ± 1.3 nm (n = 40). Apparently, such rings assemble into stacked tubular structures (seen in a side view) with a similar diameter of 10.7 ± 1.2 nm (n = 30). Stacks were variable in length ranging from 15–30 nm. Where discernible, the number of stack layers suggest a monomer height of ca. 4–5 nm. Additional structures that could not be integrated into these two categories are probably caused by insufficient focus and/or the different angles under which the structures are viewed. We interpret the stacked rings to cause the higher-order oligomers (≈ 450 kDa at GPC peak maximum) seen on native gels and upon GPC. Such large, three-dimensional arrays may represent artifacts caused by the absence of membranes, while rings may represent the functional units, bearing in mind that only higher-order homo-oligomers are enzymatically active.


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)

Electron microscopy of PDS-His6.A, negative staining. Examples of rings (white arrows) and stacks of rings (black arrows) are indicated. The inset shows examples of stacks (upper row) and rings (lower two rows) at higher magnification; each picture represents an area of 20 x 20 nm. The bar refers to the overview and represents 100 nm. B, Freeze-fracture scanning EM. Left, membrane fracture faces of liposomes containing bound PDS-His6 showing the absence of transmembrane particles. Right, membrane surfaces exposed after sublimation. The arrow points to the surface/fracture face boundary. Particles of homogenous size are seen on the surface. Bar represents 200 nm.
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Related In: Results  -  Collection

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pone.0131717.g005: Electron microscopy of PDS-His6.A, negative staining. Examples of rings (white arrows) and stacks of rings (black arrows) are indicated. The inset shows examples of stacks (upper row) and rings (lower two rows) at higher magnification; each picture represents an area of 20 x 20 nm. The bar refers to the overview and represents 100 nm. B, Freeze-fracture scanning EM. Left, membrane fracture faces of liposomes containing bound PDS-His6 showing the absence of transmembrane particles. Right, membrane surfaces exposed after sublimation. The arrow points to the surface/fracture face boundary. Particles of homogenous size are seen on the surface. Bar represents 200 nm.
Mentions: Negative staining of the higher-order oligomeric species purified in the presence of norflurazon revealed a distribution of particles, among which two recurrent patterns were observed consisting of rings (white arrows) and stacks (black arrows; Fig 5A). Rings appeared mostly four- membered, with a diameter of 11.8 ± 1.3 nm (n = 40). Apparently, such rings assemble into stacked tubular structures (seen in a side view) with a similar diameter of 10.7 ± 1.2 nm (n = 30). Stacks were variable in length ranging from 15–30 nm. Where discernible, the number of stack layers suggest a monomer height of ca. 4–5 nm. Additional structures that could not be integrated into these two categories are probably caused by insufficient focus and/or the different angles under which the structures are viewed. We interpret the stacked rings to cause the higher-order oligomers (≈ 450 kDa at GPC peak maximum) seen on native gels and upon GPC. Such large, three-dimensional arrays may represent artifacts caused by the absence of membranes, while rings may represent the functional units, bearing in mind that only higher-order homo-oligomers are enzymatically active.

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.


Related in: MedlinePlus