title: Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis.
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.
authors.affiliation: Faculty of Biology, Cell Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Gemmecker
authors.affiliation: Faculty of Biology, Cell Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Schaub
authors.affiliation: Faculty of Biology, Cell Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Koschmieder
authors.affiliation: Faculty of Chemistry and Pharmacy, Institute for Biochemistry, University of Freiburg, Freiburg, Germany.
authors.forename: Anton
authors.initials: A
authors.lastname: Brausemann
authors.affiliation: Faculty of Biology, Biochemistry and Functional Proteomics, University of Freiburg, Freiburg, Germany.
authors.forename: Friedel
authors.initials: F
authors.lastname: Drepper
authors.affiliation: Faculty of Biology, Cell Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Marta
authors.initials: M
authors.lastname: Rodriguez-Franco
authors.affiliation: Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Sandro
authors.initials: S
authors.lastname: Ghisla
authors.affiliation: Faculty of Biology, Biochemistry and Functional Proteomics, University of Freiburg, Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Warscheid
authors.affiliation: Faculty of Chemistry and Pharmacy, Institute for Biochemistry, University of Freiburg, Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Einsle
authors.affiliation: Faculty of Biology, Cell Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Beyer
mesh_terms: D001704:Biopolymers / Q000737:chemistry*; D002462:Cell Membrane / Q000201:enzymology; D018360:Crystallography, X-Ray; D013058:Mass Spectrometry; D008855:Microscopy, Electron, Scanning; D063347:Native Polyacrylamide Gel Electrophoresis; D012275:Oryza / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D001704:Biopolymers; D010088:Oxidoreductases; C022398:phytoene dehydrogenase