Structure of Phytoene Desaturase Provides Insights into Herbicide Binding and Reaction Mechanisms Involved in Carotene Desaturation.

Cyanobacteria and plants synthesize carotenoids via a poly-cis pathway starting with phytoene, a membrane-bound C40 hydrocarbon. Phytoene desaturase (PDS) introduces two double bonds and concomitantly isomerizes two neighboring double bonds from trans to cis. PDS assembles into homo-tetramers that interact monotopically with membranes. A long hydrophobic tunnel is proposed to function in the sequential binding of phytoene and the electron acceptor plastoquinone. The herbicidal inhibitor norflurazon binds at a plastoquinone site thereby blocking reoxidation of FADred. Comparison with the sequence-dissimilar bacterial carotene desaturase CRTI reveals substantial similarities in the overall protein fold, defining both as members of the GR2 family of flavoproteins. However, the PDS active center architecture is unprecedented: no functional groups are found in the immediate flavin vicinity that might participate in dehydrogenation and isomerization. This suggests that the isoalloxazine moiety is sufficient for catalysis. Despite mechanistic differences, an ancient evolutionary relation of PDS and CRTI is apparent.

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

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.

On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase.

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40) hydrocarbon substrate.