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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

Homo-oligomers of PDS-His6 analyzed by native PAGE.A, Effect of norflurazon. The high mass oligomeric fraction from GPC (purification in the absence of norflurazon in Fig 3A) dissociates into a diffuse band representing the dimer or trimer. B; the high mass oligomeric fraction from GPC separation (purification in the presence of norflurazon, Fig 3B) reveals discrete bands representing the calculated oligomeric assemblies indicated. L,M,R, left, middle and right segment of the respective high mass oligomeric GPC peaks. The corresponding low mass peaks gave a diffuse dimer/trimer when the purification was done in the absence of norflurazon and the monomer in its presence (this band has been electronically contrasted, for better visibility). Gradient gels (4–12%) cast in the absence of norflurazon containing 25 mM imidazole. C, Effect of imidazole. PDS purified in the presence of norflurazon separated on gradient gels (as above) but cast in the absence of imidazole. Lane 1, marker proteins; M, High mass GPC peak; lane 3, homo-oligomeric forms of BSA used as a standard [53]. Calculated oligomeric states of PDS-His6 are shown as boxed.
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pone.0131717.g004: Homo-oligomers of PDS-His6 analyzed by native PAGE.A, Effect of norflurazon. The high mass oligomeric fraction from GPC (purification in the absence of norflurazon in Fig 3A) dissociates into a diffuse band representing the dimer or trimer. B; the high mass oligomeric fraction from GPC separation (purification in the presence of norflurazon, Fig 3B) reveals discrete bands representing the calculated oligomeric assemblies indicated. L,M,R, left, middle and right segment of the respective high mass oligomeric GPC peaks. The corresponding low mass peaks gave a diffuse dimer/trimer when the purification was done in the absence of norflurazon and the monomer in its presence (this band has been electronically contrasted, for better visibility). Gradient gels (4–12%) cast in the absence of norflurazon containing 25 mM imidazole. C, Effect of imidazole. PDS purified in the presence of norflurazon separated on gradient gels (as above) but cast in the absence of imidazole. Lane 1, marker proteins; M, High mass GPC peak; lane 3, homo-oligomeric forms of BSA used as a standard [53]. Calculated oligomeric states of PDS-His6 are shown as boxed.

Mentions: For an analysis at higher detail than possible with GPC, PDS-His6 oligomers were investigated using non-denaturing gradient gels containing imidazole (25 mM). Fig 4A shows that the high-mass peaks isolated from GPC in the absence of norflurazon (comp. Fig 3A), produced a cloud of unresolved species plus a diffuse band corresponding to the mass of the dimer or trimer. In contrast, the high-mass peak isolated in the presence of the herbicide (comp. Fig 3B) yielded a pattern of discrete bands (Fig 4B). Semi-logarithmic regression analysis (Figure C in S1 File) suggests the presence of hepta- to undecamers, the difference in size corresponding to the monomer. Oligomers consisting of less than seven subunits were barely present.


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)

Homo-oligomers of PDS-His6 analyzed by native PAGE.A, Effect of norflurazon. The high mass oligomeric fraction from GPC (purification in the absence of norflurazon in Fig 3A) dissociates into a diffuse band representing the dimer or trimer. B; the high mass oligomeric fraction from GPC separation (purification in the presence of norflurazon, Fig 3B) reveals discrete bands representing the calculated oligomeric assemblies indicated. L,M,R, left, middle and right segment of the respective high mass oligomeric GPC peaks. The corresponding low mass peaks gave a diffuse dimer/trimer when the purification was done in the absence of norflurazon and the monomer in its presence (this band has been electronically contrasted, for better visibility). Gradient gels (4–12%) cast in the absence of norflurazon containing 25 mM imidazole. C, Effect of imidazole. PDS purified in the presence of norflurazon separated on gradient gels (as above) but cast in the absence of imidazole. Lane 1, marker proteins; M, High mass GPC peak; lane 3, homo-oligomeric forms of BSA used as a standard [53]. Calculated oligomeric states of PDS-His6 are shown as boxed.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131717.g004: Homo-oligomers of PDS-His6 analyzed by native PAGE.A, Effect of norflurazon. The high mass oligomeric fraction from GPC (purification in the absence of norflurazon in Fig 3A) dissociates into a diffuse band representing the dimer or trimer. B; the high mass oligomeric fraction from GPC separation (purification in the presence of norflurazon, Fig 3B) reveals discrete bands representing the calculated oligomeric assemblies indicated. L,M,R, left, middle and right segment of the respective high mass oligomeric GPC peaks. The corresponding low mass peaks gave a diffuse dimer/trimer when the purification was done in the absence of norflurazon and the monomer in its presence (this band has been electronically contrasted, for better visibility). Gradient gels (4–12%) cast in the absence of norflurazon containing 25 mM imidazole. C, Effect of imidazole. PDS purified in the presence of norflurazon separated on gradient gels (as above) but cast in the absence of imidazole. Lane 1, marker proteins; M, High mass GPC peak; lane 3, homo-oligomeric forms of BSA used as a standard [53]. Calculated oligomeric states of PDS-His6 are shown as boxed.
Mentions: For an analysis at higher detail than possible with GPC, PDS-His6 oligomers were investigated using non-denaturing gradient gels containing imidazole (25 mM). Fig 4A shows that the high-mass peaks isolated from GPC in the absence of norflurazon (comp. Fig 3A), produced a cloud of unresolved species plus a diffuse band corresponding to the mass of the dimer or trimer. In contrast, the high-mass peak isolated in the presence of the herbicide (comp. Fig 3B) yielded a pattern of discrete bands (Fig 4B). Semi-logarithmic regression analysis (Figure C in S1 File) suggests the presence of hepta- to undecamers, the difference in size corresponding to the monomer. Oligomers consisting of less than seven subunits were barely present.

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