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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 are enzymatically active.A, Upon GPC (Superose 6 10/300 GL column) of the IMAC-purified PDS-His6 only the high mass homo-oligomer (High) contains flavin as revealed by the concomitance of elution profile and florescence trace (orange; note that PDS-His6 fluorescence is quenched ≈ 5-fold compared to that of free FAD). B, Elution traces of PDS-His6 purified in the presence of norflurazon. C, organic extract after an incubation of the low (1) and the high mass (2) fraction in the presence of phytoene according to standard incubation conditions as defined in the Methods section. Only the high mass population converts the colorless phytoene into the pale-yellow colored ζ-carotene. D, HPLC-analysis of the assays shown in C. Lower trace, no conversion with the low mass form showing the substrates 15-cis-phytoene (1) and traces of all-trans-phytoene (2). Upper trace, of incubation with high order oligomeric PDS-His6 that shows conversion of 15-cis-phytoene (1) into cis-phytofluene (3) and cis-ζ-carotene (4). The corresponding UV-VIS spectra are shown and numbered accordingly.
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pone.0131717.g003: Homo-oligomers of PDS-His6 are enzymatically active.A, Upon GPC (Superose 6 10/300 GL column) of the IMAC-purified PDS-His6 only the high mass homo-oligomer (High) contains flavin as revealed by the concomitance of elution profile and florescence trace (orange; note that PDS-His6 fluorescence is quenched ≈ 5-fold compared to that of free FAD). B, Elution traces of PDS-His6 purified in the presence of norflurazon. C, organic extract after an incubation of the low (1) and the high mass (2) fraction in the presence of phytoene according to standard incubation conditions as defined in the Methods section. Only the high mass population converts the colorless phytoene into the pale-yellow colored ζ-carotene. D, HPLC-analysis of the assays shown in C. Lower trace, no conversion with the low mass form showing the substrates 15-cis-phytoene (1) and traces of all-trans-phytoene (2). Upper trace, of incubation with high order oligomeric PDS-His6 that shows conversion of 15-cis-phytoene (1) into cis-phytofluene (3) and cis-ζ-carotene (4). The corresponding UV-VIS spectra are shown and numbered accordingly.

Mentions: The oligomeric state of PDS-His6 was investigated using calibrated HiLoad Superdex 200 and Superose 6 10/300 GL columns (5–1000 kDa and 10–600 kDa separation ranges, respectively). In both cases (Fig 3A), two distinct populations of PDS-His6 eluted, corresponding to ≈ 56 kDa and to ≈ 450 kDa, at peak maximum. These can be assigned to the monomeric (calculated mass: 56.2 kDa) form and a population around the octameric form of the enzyme (Fig A in S1 File). Fluorescence traces recorded in parallel and photometric quantification of the flavin released after heat-denaturation are consistent with the assumption that the presumed octameric form contains approximately stoichiometric equivalents of flavin, while flavin association was much lower in the low-mass form. Accordingly, specific activities correlated with the ≈ 450 kDa elution peak, while only residual activity was detected in the low-mass peak, corresponding to ≤ 1.3% of that of the octameric form. Incubation of the high-mass form in the presence of phytoene-containing liposomes and decylplastoquinone resulted in the formation of yellowish ζ-carotene (Fig 3C). An incubation experiment showed a typical conversion rate of 6 nmol min-1 mg-1, resulting in 21% conversion of 15-cis-phytoene after 10 min. HPLC analysis of the extract showed the appearance of the intermediate 9, 15-di-cis-phytofluene, in addition to the final product 9, 15, 9´-tri-cis-ζ-carotene.


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 are enzymatically active.A, Upon GPC (Superose 6 10/300 GL column) of the IMAC-purified PDS-His6 only the high mass homo-oligomer (High) contains flavin as revealed by the concomitance of elution profile and florescence trace (orange; note that PDS-His6 fluorescence is quenched ≈ 5-fold compared to that of free FAD). B, Elution traces of PDS-His6 purified in the presence of norflurazon. C, organic extract after an incubation of the low (1) and the high mass (2) fraction in the presence of phytoene according to standard incubation conditions as defined in the Methods section. Only the high mass population converts the colorless phytoene into the pale-yellow colored ζ-carotene. D, HPLC-analysis of the assays shown in C. Lower trace, no conversion with the low mass form showing the substrates 15-cis-phytoene (1) and traces of all-trans-phytoene (2). Upper trace, of incubation with high order oligomeric PDS-His6 that shows conversion of 15-cis-phytoene (1) into cis-phytofluene (3) and cis-ζ-carotene (4). The corresponding UV-VIS spectra are shown and numbered accordingly.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131717.g003: Homo-oligomers of PDS-His6 are enzymatically active.A, Upon GPC (Superose 6 10/300 GL column) of the IMAC-purified PDS-His6 only the high mass homo-oligomer (High) contains flavin as revealed by the concomitance of elution profile and florescence trace (orange; note that PDS-His6 fluorescence is quenched ≈ 5-fold compared to that of free FAD). B, Elution traces of PDS-His6 purified in the presence of norflurazon. C, organic extract after an incubation of the low (1) and the high mass (2) fraction in the presence of phytoene according to standard incubation conditions as defined in the Methods section. Only the high mass population converts the colorless phytoene into the pale-yellow colored ζ-carotene. D, HPLC-analysis of the assays shown in C. Lower trace, no conversion with the low mass form showing the substrates 15-cis-phytoene (1) and traces of all-trans-phytoene (2). Upper trace, of incubation with high order oligomeric PDS-His6 that shows conversion of 15-cis-phytoene (1) into cis-phytofluene (3) and cis-ζ-carotene (4). The corresponding UV-VIS spectra are shown and numbered accordingly.
Mentions: The oligomeric state of PDS-His6 was investigated using calibrated HiLoad Superdex 200 and Superose 6 10/300 GL columns (5–1000 kDa and 10–600 kDa separation ranges, respectively). In both cases (Fig 3A), two distinct populations of PDS-His6 eluted, corresponding to ≈ 56 kDa and to ≈ 450 kDa, at peak maximum. These can be assigned to the monomeric (calculated mass: 56.2 kDa) form and a population around the octameric form of the enzyme (Fig A in S1 File). Fluorescence traces recorded in parallel and photometric quantification of the flavin released after heat-denaturation are consistent with the assumption that the presumed octameric form contains approximately stoichiometric equivalents of flavin, while flavin association was much lower in the low-mass form. Accordingly, specific activities correlated with the ≈ 450 kDa elution peak, while only residual activity was detected in the low-mass peak, corresponding to ≤ 1.3% of that of the octameric form. Incubation of the high-mass form in the presence of phytoene-containing liposomes and decylplastoquinone resulted in the formation of yellowish ζ-carotene (Fig 3C). An incubation experiment showed a typical conversion rate of 6 nmol min-1 mg-1, resulting in 21% conversion of 15-cis-phytoene after 10 min. HPLC analysis of the extract showed the appearance of the intermediate 9, 15-di-cis-phytofluene, in addition to the final product 9, 15, 9´-tri-cis-ζ-carotene.

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