Plant-type phytoene desaturase: Functional evaluation of structural implications
Koschmieder J, Fehling-Kaschek M, Schaub P, Ghisla S, Brausemann A, Timmer J, Beyer P.
Phytoene desaturase (PDS) is an essential carotenoid biosynthetic enzyme in plants and a prominent target of certain inhibitors, such as norflurazon, acting as bleaching herbicides. PDS catalyzes the introduction of two double bonds into 15-cis-phytoene, yielding 9,15,9 ́-tri-cis-ζ-carotene via the intermediate 9,15-di-cis-phytofluene. We here present the necessary data to scrutinize functional implications inferred from the recently resolved structure of PDS in a complex with norflurazon. Using dynamic mathematical modeling of time courses, we support the relevance of the homotetrameric assembly of the enzyme in crystallo by providing evidence for substrate channeling of the intermediate phytofluene between individual subunits at membrane surfaces. Kinetic investigations support an ordered ping pong bi-bi mechanism in which the carotene and the quinone electron acceptor successively occupy the same catalytic site. Norflurazon competes with plastoquinone and mutagenesis of a conserved arginine residue corroborates the possibility of engineering herbicide resistance, however, at the expense of diminished catalytic activity. These data also support a “flavin only” mechanism of carotene desaturation not requiring charged residues in the active site. Finally, evidence for the role of the central phytoene cis double bond in determining the regio-specificity of carotene desaturation is presented.
