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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

Membrane association and chemical cross-linking of PDS-His6.A, SDS-PAGE (12%, Coomassie Blue-stained) analysis of liposomal binding assays. Lanes 1 and 2; 50% of the PDS-His6 amount added to the liposomal suspension (25 μg). Lanes 3 and 4 represent 100% of the liposome-bound PDS-His6, after centrifugation onto a 30% sucrose cushion. Equal band strengths observed thus indicate ca. 50% protein binding. These liposomes were washed either with incubation buffer (lane 5) or 0.5 M KCl in incubation buffer (lane 6) and recollected by ultracentrifugation. 100% of this material was applied. The sample duplication represents independent experiments. B, SDS-PAGE (12%, silver stained) of cross-linked PDS-His6 collected from the high mass oligomeric GPC peak obtained in the absence of norflurazon (as shown in Fig 3A). Lanes 1, 2, untreated PDS; M, marker proteins. The chemical cross linkers used were DSS (lane 3), DSG (lane 4), DSP (lane 5) and TSAT (lane 6). Dimeric reaction products were predominantly formed (boxed).
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pone.0131717.g006: Membrane association and chemical cross-linking of PDS-His6.A, SDS-PAGE (12%, Coomassie Blue-stained) analysis of liposomal binding assays. Lanes 1 and 2; 50% of the PDS-His6 amount added to the liposomal suspension (25 μg). Lanes 3 and 4 represent 100% of the liposome-bound PDS-His6, after centrifugation onto a 30% sucrose cushion. Equal band strengths observed thus indicate ca. 50% protein binding. These liposomes were washed either with incubation buffer (lane 5) or 0.5 M KCl in incubation buffer (lane 6) and recollected by ultracentrifugation. 100% of this material was applied. The sample duplication represents independent experiments. B, SDS-PAGE (12%, silver stained) of cross-linked PDS-His6 collected from the high mass oligomeric GPC peak obtained in the absence of norflurazon (as shown in Fig 3A). Lanes 1, 2, untreated PDS; M, marker proteins. The chemical cross linkers used were DSS (lane 3), DSG (lane 4), DSP (lane 5) and TSAT (lane 6). Dimeric reaction products were predominantly formed (boxed).

Mentions: PDS-His6, although purified as a soluble protein, must be able to interact with membranes since the carotene substrate provided in liposomal membranes resides within the hydrophobic core [38]. In fact, the protein binds spontaneously to liposomal membranes (Fig 6A). Ultracentrifugation of liposomes onto a 30% sucrose cushion after addition of PDS-His6 showed ≈ 50% protein recovery in the liposome band. High-salt (0.5 M KCl) treatment of these isolated liposomes followed by additional centrifugation revealed that the interaction is apparently hydrophobic, since the protein was retained. The somewhat weaker bands observed are probably due to certain losses of liposomes caused by the repeated centrifugation and collection.


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)

Membrane association and chemical cross-linking of PDS-His6.A, SDS-PAGE (12%, Coomassie Blue-stained) analysis of liposomal binding assays. Lanes 1 and 2; 50% of the PDS-His6 amount added to the liposomal suspension (25 μg). Lanes 3 and 4 represent 100% of the liposome-bound PDS-His6, after centrifugation onto a 30% sucrose cushion. Equal band strengths observed thus indicate ca. 50% protein binding. These liposomes were washed either with incubation buffer (lane 5) or 0.5 M KCl in incubation buffer (lane 6) and recollected by ultracentrifugation. 100% of this material was applied. The sample duplication represents independent experiments. B, SDS-PAGE (12%, silver stained) of cross-linked PDS-His6 collected from the high mass oligomeric GPC peak obtained in the absence of norflurazon (as shown in Fig 3A). Lanes 1, 2, untreated PDS; M, marker proteins. The chemical cross linkers used were DSS (lane 3), DSG (lane 4), DSP (lane 5) and TSAT (lane 6). Dimeric reaction products were predominantly formed (boxed).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131717.g006: Membrane association and chemical cross-linking of PDS-His6.A, SDS-PAGE (12%, Coomassie Blue-stained) analysis of liposomal binding assays. Lanes 1 and 2; 50% of the PDS-His6 amount added to the liposomal suspension (25 μg). Lanes 3 and 4 represent 100% of the liposome-bound PDS-His6, after centrifugation onto a 30% sucrose cushion. Equal band strengths observed thus indicate ca. 50% protein binding. These liposomes were washed either with incubation buffer (lane 5) or 0.5 M KCl in incubation buffer (lane 6) and recollected by ultracentrifugation. 100% of this material was applied. The sample duplication represents independent experiments. B, SDS-PAGE (12%, silver stained) of cross-linked PDS-His6 collected from the high mass oligomeric GPC peak obtained in the absence of norflurazon (as shown in Fig 3A). Lanes 1, 2, untreated PDS; M, marker proteins. The chemical cross linkers used were DSS (lane 3), DSG (lane 4), DSP (lane 5) and TSAT (lane 6). Dimeric reaction products were predominantly formed (boxed).
Mentions: PDS-His6, although purified as a soluble protein, must be able to interact with membranes since the carotene substrate provided in liposomal membranes resides within the hydrophobic core [38]. In fact, the protein binds spontaneously to liposomal membranes (Fig 6A). Ultracentrifugation of liposomes onto a 30% sucrose cushion after addition of PDS-His6 showed ≈ 50% protein recovery in the liposome band. High-salt (0.5 M KCl) treatment of these isolated liposomes followed by additional centrifugation revealed that the interaction is apparently hydrophobic, since the protein was retained. The somewhat weaker bands observed are probably due to certain losses of liposomes caused by the repeated centrifugation and collection.

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