Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis

• Published in: PloS one Vol. 10; no. 7; p. e0131717
• Main Authors: Gemmecker, Sandra, Schaub, Patrick, Koschmieder, Julian, Brausemann, Anton, Drepper, Friedel, Rodriguez-Franco, Marta, Ghisla, Sandro, Warscheid, Bettina, Einsle, Oliver, Beyer, Peter
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 06.07.2015; Public Library of Science (PLoS)
• Subjects: Amino acid sequence; Antifungal agents; Arabidopsis; Bacteria; Binding sites; Biochemistry; Biology; Biopolymers - chemistry; Biosynthesis; Blockage; Carotene; Carotenoids; Cell Membrane - enzymology; Chloroplasts; Chromatography; Crosslinking; Crystallography, X-Ray; Desaturase; Desaturation; Dimers; Electron microscopy; Flavin-adenine dinucleotide; Flavoproteins; Homogeneity; Homology; Liquid chromatography; Mass Spectrometry; Mass spectroscopy; Microscopy, Electron, Scanning; Mutation; Native Polyacrylamide Gel Electrophoresis; Norflurazon; Oligomers; Oryza - enzymology; Oxidoreductase; Oxidoreductases - chemistry; Oxidoreductases - isolation & purification; Oxidoreductases - metabolism; Oxygen; Pharmacy; Plastids; Protein Binding; Protein Conformation; Protein folding; Proteins; Proteomics; Quinone; Quinones; Structural analysis
• DOI: 10.1371/journal.pone.0131717
• ISSN: 1932-6203

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.