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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.


The PDS/ZDS carotene desaturation sequence.Magenta double bonds; introduced in trans configuration, green double bonds introduced in cis configuration. Blue arrows show isomerization from trans to cis, black arrows from cis to trans. Redox pathways in chloroplasts employ photosynthetic electron transport while a route through PTOX takes place in developing chloroplasts and non-green plastids. Both routes may as well prevail with ZDS. For further explanations, see text.
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pone.0131717.g001: The PDS/ZDS carotene desaturation sequence.Magenta double bonds; introduced in trans configuration, green double bonds introduced in cis configuration. Blue arrows show isomerization from trans to cis, black arrows from cis to trans. Redox pathways in chloroplasts employ photosynthetic electron transport while a route through PTOX takes place in developing chloroplasts and non-green plastids. Both routes may as well prevail with ZDS. For further explanations, see text.

Mentions: Phytoene desaturases (PDS), the subject of this work, prevail in plants and cyanobacteria; they introduce two double bonds into the symmetric, colorless phytoene substrate. This extends the triene chromophore of phytoene to form—via the pentaene intermediate phytofluene—the light yellow ζ-carotene with seven conjugated double bonds (Fig 1). The new double bonds are inserted symmetrically at positions C11 and C11´. A second, homologous desaturase, ζ-carotene desaturase (ZDS) is required to insert two additional double bonds at positions C7 and C7´. This leads to the red-colored lycopene with 11 conjugated double bonds.


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)

The PDS/ZDS carotene desaturation sequence.Magenta double bonds; introduced in trans configuration, green double bonds introduced in cis configuration. Blue arrows show isomerization from trans to cis, black arrows from cis to trans. Redox pathways in chloroplasts employ photosynthetic electron transport while a route through PTOX takes place in developing chloroplasts and non-green plastids. Both routes may as well prevail with ZDS. For further explanations, see text.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131717.g001: The PDS/ZDS carotene desaturation sequence.Magenta double bonds; introduced in trans configuration, green double bonds introduced in cis configuration. Blue arrows show isomerization from trans to cis, black arrows from cis to trans. Redox pathways in chloroplasts employ photosynthetic electron transport while a route through PTOX takes place in developing chloroplasts and non-green plastids. Both routes may as well prevail with ZDS. For further explanations, see text.
Mentions: Phytoene desaturases (PDS), the subject of this work, prevail in plants and cyanobacteria; they introduce two double bonds into the symmetric, colorless phytoene substrate. This extends the triene chromophore of phytoene to form—via the pentaene intermediate phytofluene—the light yellow ζ-carotene with seven conjugated double bonds (Fig 1). The new double bonds are inserted symmetrically at positions C11 and C11´. A second, homologous desaturase, ζ-carotene desaturase (ZDS) is required to insert two additional double bonds at positions C7 and C7´. This leads to the red-colored lycopene with 11 conjugated double bonds.

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.