Fig 1.
(A) The symmetrical substrate, 15-cis-phytoene is desaturated twice at the symmetrical positions indicated in magenta. The simultaneous isomerization of the adjacent double bonds (arrows) from trans to cis yields the symmetric product 9,15,9'-tri-cis-ζ-carotene via the asymmetric intermediate, 9,15-di-cis-phytofluene. Electrons are transferred from the reduced enzyme-bound FAD onto the terminal electron acceptor plastoquinone which is reoxidized by the photosynthetic electron transport chain or, alternatively, by the plastid terminal oxidase PTOX (sequence omitted in the second partial reaction). (B) Overview on the tetrameric PDS assembly as viewed from the plane of the membrane. The substrate entry channels are outlined in blue, FAD is represented as sticks and balls and highlighted in yellow, norflurazon is represented as green sticks.
Fig 2.
Basic characterization of the PDS reaction.
Dependency of the PDS reaction rate on protein concentration (A), pH (B) and temperature (C) and reaction time course of phytofluene and ζ-carotene formation from phytoene (D). ▼, phytoene; ○, phytofluene; ■, ζ-carotene. Each experiment (A-C) was carried out using the optimum values of the respective non-variable parameters e.g. pH 6.0, 37°C in A, etc. The optimal values obtained defined the standard incubation conditions (see Methods). The standard protein concentration was set to 25 μg PDS per assay. [p] = 10 mM, [DPQ] = 19.25 mM, as determined elsewhere (see Fig 6). The samples were analyzed by HPLC after an incubation time of 10 min. Data represent the mean of duplicates (A, C) or triplicates (B) ± SEM. D, Asterisks denote the activation of phytofluene and ζ-carotene formation upon the addition of fresh PDS during the plateau phase after 30 min. Data were fitted with splines in A, C and D and with the dibasic pH equation (see Methods) in B.
Fig 3.
Stereoconfiguration of PDS products.
(A) Phytofluene isomers: trace a represents phytofluene from a PDS assay. The peak marked with * represents the ζ-carotene formed. Only the correct 9,15-di-cis-phytofluene isomer is formed as revealed by comparison with authentic standards isolated from sources where cis-configurations are known, such as trace b, phytofluene from the tangerine mutant of tomato fruit [31] and trace c, phytofluene from Dunaliella bardawil grown in the presence of norflurazon [32]. The synthetic standards all-trans and 15-cis-phytofluene are shown in trace d. (B) ζ-carotene isomers: trace e, from PDS assays. Only the correct 9,15,9’-tri-cis-ζ-carotene is formed, as revealed by the effect of illumination of the PDS assay (trace f) whereby the photolabile central double bond is isomerized to trans [4, 24] yielding the 9,9’-di-cis species accompanied by small amounts of the 9-cis and all-trans species. Trace g, extract from tangerine tomato fruit containing 9,9’-di-cis-ζ-carotene. The peak marked with * represents β-carotene, detected because of spectral overlap. HPLC traces (HPLC system 2) were recorded at 400 nm. UV/VIS spectra are given as insets.
Fig 4.
Kinetic scheme of the monomeric model and dynamic modeling of PDS reaction time courses.
(A) Monomeric model. PDS monomeric subunits (orange and blue rectangles) within the homotetramer are assumed to work independently. Orange/blue color denotes reduced/oxidized half sides of phytoene (p), phytofluene (pf) and ζ-carotene (z) and the respective redox state of the PDS-bound FAD. The overall reaction comprises the three main processes phytoene desaturation (i), phytofluene desaturation (ii) and plastoquinone reduction (iii) with the rate constants kp, kpf and krox, respectively. Each rate constant encompasses the three equilibria represented by the reaction arrows associated to each of the three main processes which are highlighted by shadowed areas: association-dissociation of enzyme and substrate, desaturation-saturation of substrate and dissociation-association of enzyme and product. All hydrophobic carotene substrates and DPQ (Q) are soluble in the hydrophobic core of liposomal membranes. Progressive inactivation of PDS by denaturation (iv) is a process to be considered. (B-D) Reaction time courses of phytoene and phytofluene conversion by PDS. Reaction time courses were initiated [p] = 3.7 nmol (p high; B), [p] = 1.3 nmol (p low; C) and [pf] = 5.2 nmol (pf; D). The observables are given as data points (black, phytoene, p; red, phytofluene, pf; blue, ζ-carotene, pf), the model fit (obtained with model I; ODE 1–5) is represented by lines. The modeling was either based on simultaneous parameter estimation for all three reaction time courses (solid lines) or on simultaneous estimation of kp, krox and kage and individual estimation of kpf (dashed line). Measurements were carried out in triplicate.
Table 1.
Parameter values for the monomeric and the substrate channeling model.
Fig 5.
Kinetic scheme of the substrate channeling model and dynamic modeling of PDS reaction time courses.
(A) Substrate channeling model, accounting for substrate channeling between PDS homotetramers. Symbols are as given in Fig 4A. Two species of phytofluene, i.e. phytofluene fates, coexist. Left; nascent phytofluene (pf*) that is produced from phytoene (p) can be restricted in its diffusion into the membrane residing in a microdomain in proximity to the PDS homotetramer, as indicated by the bent arrow. It can be channeled into a second PDS subunit of the homotetramer containing FADox, allowing rapid conversion to ζ-carotene (z) with the rate constant kpf*. Right; pf* can alternatively diffuse into PDS-distant membrane areas with rate constant kdiff, this defining the species pf. From there it can be taken up by another monomeric PDS subunit and be converted into ζ-carotene (z) with rate constant kpf. Rate constant kage represents enzyme inactivation which refers to both the reduced and oxidized enzyme states. (B-G) Dynamic modeling of reaction time courses of phytoene and phytofluene conversion by PDS. Reaction time courses were conducted with 1.3 nmol phytoene (p low; B), and 3.7 nmol phytoene (p high; C). In addition, liposomes containing 5.2 nmol phytofluene were used (pf; D). The observables are given as data points (black, phytoene, p; red, phytofluene, pf; blue, ζ-carotene, z). The model fit, represented by lines, is based on Eqs 1 and 6–10 with simultaneous parameter estimation for all three reaction time courses. Shadowed areas indicate one standard deviation as estimated by the error model (see Methods). Measurements were carried out in triplicate. (E) Prediction of the amount of oxidized, active PDS (ox) and reduced PDS (red) over time, indicating a rapid decrease in oxidized and reduced PDS levels due to enzyme inactivation. (F,G) Deduced carotene fluxes through the different sub-processes labeled with their rate constants (see Fig 4). Note the different scaling in F and G. Flux predictions are based on the phytoene conversion reaction time course “p high” (C).
Fig 6.
Data and model predictions on concentration-dependent PDS reaction rates.
Measured (A-C) and simulated (D-E) concentration dependency of the PDS reaction rates. Dependency on (A) DPQ determined at [p] = 40 mM (≈ 1 x KM), (B) phytoene measured at [DPQ] = 19.25 mM (≈ 15 x KM) and (C) phytofluene measured at [DPQ] = 19.25 mM. Data represent triplicates ± SEM. Phytofluene and ζ-carotene formation in A–C were fitted with the MM equation (see Methods; solid lines; goodness of fit for ζ-carotene formation: A, R2 = 0.98; B, R2 = 0.97; C, R2 = 0.98) except phytofluene formation in B that was fitted with a spline. The ζ-carotene:phytofluene ratios in A and B are given as dotted lines and plotted to the right y-axis. Date are given as squares the solid lines represent the fit (A-C) or model prediction (D-F). Red color denotes ζ-carotene; blue represents phytofluene. Shadowed areas in D—F represent one standard deviation.
Table 2.
Observed and estimated apparent KM and Vmax values for PDS substrates.
Fig 7.
DPQ and phytoene concentration dependencies of PDS inhibition by NFZ.
PDS inhibition was investigated at the indicated increasing concentrations of the inhibitor NFZ and of the substrates (A), DPQ and (B), phytoene. Data represent triplicates ± SEM and were fitted with the equation for competitive inhibition (A; R2 = 0.99) and the Michaelis-Menten equation (B; 0.95) using the GraphPad Prism 5 software. Data obtained in the presence of NFZ in B were not fitted due to poor goodness of fit with the equations for competitive, non-competitive and uncompetitive inhibition (for equations, see Methods). All other assay parameters were as defined (for standard conditions, see Methods).
Fig 8.
Kinetic characterization of Arg300Ser PDS.
Dependence of Arg300Ser PDS reaction rates on phytoene (A) and DPQ (B). (C) Inhibition kinetics of Arg300Ser PDS in a matrix of varying DPQ and NFZ concentration. Data points represent the mean of duplicates ± SEM. In A and B: ■, ζ-carotene; ○, phytofluene; Δ, ζ-carotene:phytofluene ratio. Data in A (R2 = 0.58) and B (R2 = 0.95) were fitted with the Michaelis-Menten equation and the equation for competitive inhibition was applied in C (R2 = 0.92) using the GraphPad Prism 5 software (for equations, see Methods). The product:intermediate ratios in A and B (dotted lines; plotted to the right Y-axis) was fitted using a spline. Assays were carried out under standard conditions and incubated for 15 min (see Methods).
Fig 9.
LC-MS analysis of PDS desaturation products produced from asymmetric (C35) 15-cis-nor-phytoene.
(A) Structure of 15-cis-1',2',3',16',17'-penta-nor-phytoene (15-cis-nor-phytoene). The desaturation sites C11-C12 and C11’-C12’ and the central C15-C15’ double bond are marked. The carbon bonds located above the redox-reactive isoalloxazine are indicated by arrows if substrate positioning is mediated by the central 15-cis-configured triene (I) or substrate cavity back end (II). See text for details. (B) Identification of PDS desaturation products by LC-MS analysis. Carotenes were detected photometrically in the 275–400 nm range (top panel). The UV/VIS spectra of 15-cis-nor-phytoene and the desaturation products are shown (central panel). The bottom panel shows the corresponding MS1 spectra with the exact masses of the quasi-molecular ions [M+H]+, the derived sum formula and the mass deviation.