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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

Effect of the redox state of FAD and of norfluorazon on the stability of PDS-His6.Thermo-FAD measurements were carried out in a RealTime-PCR instrument using the SYBR-Green Channel (Exc = 488 nm; Em = 520 nm). Trace 1; PDS-His6 isolated in the absence of norflurazon containing FADox. Trace 2; PDS-His6 isolated in the absence of norflurazon containing FADox after addition of 50 μM norflurazon, Trace 3; PDS isolated in the presence of 50 μM norflurazon containing FADred.
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pone.0131717.g009: Effect of the redox state of FAD and of norfluorazon on the stability of PDS-His6.Thermo-FAD measurements were carried out in a RealTime-PCR instrument using the SYBR-Green Channel (Exc = 488 nm; Em = 520 nm). Trace 1; PDS-His6 isolated in the absence of norflurazon containing FADox. Trace 2; PDS-His6 isolated in the absence of norflurazon containing FADox after addition of 50 μM norflurazon, Trace 3; PDS isolated in the presence of 50 μM norflurazon containing FADred.

Mentions: Alterations of FAD fluorescence can be used to monitor (un)folding processes of flavoproteins in response to temperature [39]. Fig 9 shows such studies with PDS-His6, purified in the absence of norflurazon therefore containing FADox (Fig 6A, curve 1). The increase in fluorescence shows an inflection point at 44°C, reflecting denaturation. Addition of norflurazon to the oxidized protein modifies this response towards a more biphasic behavior accompanied and by a small increase in thermostability (curve 2). A drastic increase in thermostability of ≈ 22°C is observed with PDS-His6 that was isolated in the continuous presence of norflurazon and thus contained reduced FAD (curve 3). This can point towards a substantial structural change of the enzyme in response to the redox state of the flavin.


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)

Effect of the redox state of FAD and of norfluorazon on the stability of PDS-His6.Thermo-FAD measurements were carried out in a RealTime-PCR instrument using the SYBR-Green Channel (Exc = 488 nm; Em = 520 nm). Trace 1; PDS-His6 isolated in the absence of norflurazon containing FADox. Trace 2; PDS-His6 isolated in the absence of norflurazon containing FADox after addition of 50 μM norflurazon, Trace 3; PDS isolated in the presence of 50 μM norflurazon containing FADred.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131717.g009: Effect of the redox state of FAD and of norfluorazon on the stability of PDS-His6.Thermo-FAD measurements were carried out in a RealTime-PCR instrument using the SYBR-Green Channel (Exc = 488 nm; Em = 520 nm). Trace 1; PDS-His6 isolated in the absence of norflurazon containing FADox. Trace 2; PDS-His6 isolated in the absence of norflurazon containing FADox after addition of 50 μM norflurazon, Trace 3; PDS isolated in the presence of 50 μM norflurazon containing FADred.
Mentions: Alterations of FAD fluorescence can be used to monitor (un)folding processes of flavoproteins in response to temperature [39]. Fig 9 shows such studies with PDS-His6, purified in the absence of norflurazon therefore containing FADox (Fig 6A, curve 1). The increase in fluorescence shows an inflection point at 44°C, reflecting denaturation. Addition of norflurazon to the oxidized protein modifies this response towards a more biphasic behavior accompanied and by a small increase in thermostability (curve 2). A drastic increase in thermostability of ≈ 22°C is observed with PDS-His6 that was isolated in the continuous presence of norflurazon and thus contained reduced FAD (curve 3). This can point towards a substantial structural change of the enzyme in response to the redox state of the flavin.

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