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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.


Purification of PDS-His6.SDS-PAGE (10%; stain Coomassie Blue) showing in lane 1, E. coli lysate after centrifugation at 18,600 x g; Lane 2, PDS-His6 after IMAC purification; lane 3, PDS-His6 after GPC purification; M, MW protein standards.
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pone.0131717.g002: Purification of PDS-His6.SDS-PAGE (10%; stain Coomassie Blue) showing in lane 1, E. coli lysate after centrifugation at 18,600 x g; Lane 2, PDS-His6 after IMAC purification; lane 3, PDS-His6 after GPC purification; M, MW protein standards.

Mentions: The present PDS-His6 purification protocol is the result of a substantial optimization required to overcome the notorious tendency of the protein for aggregation. The detergent CHAPS at concentrations well below its CMC was found to be best suited for detaching the protein from E. coli membranes while, surprisingly, no supplementation of detergents was required during all subsequent steps. The SDS-PAGE analysis depicted in Fig 2 documents a ≈ 4000-fold enrichment of PDS-His6 that was achieved primarily during the IMAC step (lane 2). Most of the residual contaminants were removed by GPC (lane 3), during which the presence of 150 mM imidazole was crucial. In its absence most PDS-His6 eluted as aggregates in the dead volume and is almost completely lost by adsorption upon ultrafiltration. On the downside, imidazole severely decreased the stability of the protein and inhibited its activity; it therefore had to be dialyzed off for both purposes. Concentrated PDS-His6 solutions are yellow, consistent with the presence of a flavin cofactor.


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)

Purification of PDS-His6.SDS-PAGE (10%; stain Coomassie Blue) showing in lane 1, E. coli lysate after centrifugation at 18,600 x g; Lane 2, PDS-His6 after IMAC purification; lane 3, PDS-His6 after GPC purification; M, MW protein standards.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131717.g002: Purification of PDS-His6.SDS-PAGE (10%; stain Coomassie Blue) showing in lane 1, E. coli lysate after centrifugation at 18,600 x g; Lane 2, PDS-His6 after IMAC purification; lane 3, PDS-His6 after GPC purification; M, MW protein standards.
Mentions: The present PDS-His6 purification protocol is the result of a substantial optimization required to overcome the notorious tendency of the protein for aggregation. The detergent CHAPS at concentrations well below its CMC was found to be best suited for detaching the protein from E. coli membranes while, surprisingly, no supplementation of detergents was required during all subsequent steps. The SDS-PAGE analysis depicted in Fig 2 documents a ≈ 4000-fold enrichment of PDS-His6 that was achieved primarily during the IMAC step (lane 2). Most of the residual contaminants were removed by GPC (lane 3), during which the presence of 150 mM imidazole was crucial. In its absence most PDS-His6 eluted as aggregates in the dead volume and is almost completely lost by adsorption upon ultrafiltration. On the downside, imidazole severely decreased the stability of the protein and inhibited its activity; it therefore had to be dialyzed off for both purposes. Concentrated PDS-His6 solutions are yellow, consistent with the presence of a flavin cofactor.

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.