Fig 1.
The PDS/ZDS carotene desaturation sequence.
Magenta double bonds; introduced in trans configuration, green double bonds introduced in cis configuration. Blue arrows show isomerization from trans to cis, black arrows from cis to trans. Redox pathways in chloroplasts employ photosynthetic electron transport while a route through PTOX takes place in developing chloroplasts and non-green plastids. Both routes may as well prevail with ZDS. For further explanations, see text.
Fig 2.
SDS-PAGE (10%; stain Coomassie Blue) showing in lane 1, E. coli lysate after centrifugation at 18,600 x g; Lane 2, PDS-His6 after IMAC purification; lane 3, PDS-His6 after GPC purification; M, MW protein standards.
Fig 3.
Homo-oligomers of PDS-His6 are enzymatically active.
A, Upon GPC (Superose 6 10/300 GL column) of the IMAC-purified PDS-His6 only the high mass homo-oligomer (High) contains flavin as revealed by the concomitance of elution profile and florescence trace (orange; note that PDS-His6 fluorescence is quenched ≈ 5-fold compared to that of free FAD). B, Elution traces of PDS-His6 purified in the presence of norflurazon. C, organic extract after an incubation of the low (1) and the high mass (2) fraction in the presence of phytoene according to standard incubation conditions as defined in the Methods section. Only the high mass population converts the colorless phytoene into the pale-yellow colored ζ-carotene. D, HPLC-analysis of the assays shown in C. Lower trace, no conversion with the low mass form showing the substrates 15-cis-phytoene (1) and traces of all-trans-phytoene (2). Upper trace, of incubation with high order oligomeric PDS-His6 that shows conversion of 15-cis-phytoene (1) into cis-phytofluene (3) and cis-ζ-carotene (4). The corresponding UV-VIS spectra are shown and numbered accordingly.
Fig 4.
Homo-oligomers of PDS-His6 analyzed by native PAGE.
A, Effect of norflurazon. The high mass oligomeric fraction from GPC (purification in the absence of norflurazon in Fig 3A) dissociates into a diffuse band representing the dimer or trimer. B; the high mass oligomeric fraction from GPC separation (purification in the presence of norflurazon, Fig 3B) reveals discrete bands representing the calculated oligomeric assemblies indicated. L,M,R, left, middle and right segment of the respective high mass oligomeric GPC peaks. The corresponding low mass peaks gave a diffuse dimer/trimer when the purification was done in the absence of norflurazon and the monomer in its presence (this band has been electronically contrasted, for better visibility). Gradient gels (4–12%) cast in the absence of norflurazon containing 25 mM imidazole. C, Effect of imidazole. PDS purified in the presence of norflurazon separated on gradient gels (as above) but cast in the absence of imidazole. Lane 1, marker proteins; M, High mass GPC peak; lane 3, homo-oligomeric forms of BSA used as a standard [53]. Calculated oligomeric states of PDS-His6 are shown as boxed.
Fig 5.
Electron microscopy of PDS-His6.
A, negative staining. Examples of rings (white arrows) and stacks of rings (black arrows) are indicated. The inset shows examples of stacks (upper row) and rings (lower two rows) at higher magnification; each picture represents an area of 20 x 20 nm. The bar refers to the overview and represents 100 nm. B, Freeze-fracture scanning EM. Left, membrane fracture faces of liposomes containing bound PDS-His6 showing the absence of transmembrane particles. Right, membrane surfaces exposed after sublimation. The arrow points to the surface/fracture face boundary. Particles of homogenous size are seen on the surface. Bar represents 200 nm.
Fig 6.
Membrane association and chemical cross-linking of PDS-His6.
A, SDS-PAGE (12%, Coomassie Blue-stained) analysis of liposomal binding assays. Lanes 1 and 2; 50% of the PDS-His6 amount added to the liposomal suspension (25 μg). Lanes 3 and 4 represent 100% of the liposome-bound PDS-His6, after centrifugation onto a 30% sucrose cushion. Equal band strengths observed thus indicate ca. 50% protein binding. These liposomes were washed either with incubation buffer (lane 5) or 0.5 M KCl in incubation buffer (lane 6) and recollected by ultracentrifugation. 100% of this material was applied. The sample duplication represents independent experiments. B, SDS-PAGE (12%, silver stained) of cross-linked PDS-His6 collected from the high mass oligomeric GPC peak obtained in the absence of norflurazon (as shown in Fig 3A). Lanes 1, 2, untreated PDS; M, marker proteins. The chemical cross linkers used were DSS (lane 3), DSG (lane 4), DSP (lane 5) and TSAT (lane 6). Dimeric reaction products were predominantly formed (boxed).
Table 1.
Cross-linked peptides of phytoene desaturase identified by mass spectrometry.
Fig 7.
Preliminary structural analysis of PDS-His6 by X-ray crystallography.
A, Stereo representation of a representative section of the experimental electron density map as a blue mesh, with initial α-helical models shown as cylinders. B, Superposition of a preliminary PDS-model shown as yellow cylinders with the structure of CRTI (PDB-ID: 4DGK) in blue, displaying the high similarity in the overall structure of the two enzymes. C; Cartoon representation of a superposition of CRTI in blue with an oxidoreductase from Methanosarcina mazei (PDB-ID: 3KA7) in grey and its FAD cofactor in yellow.
Table 2.
Data collection statistics.
Fig 8.
Spectral properties of PDS and roles of quinones and of norflurazon.
A Trace; UV-VIS spectra of PDS-His6 that was purified in the presence of norflurazon. The low absorbance in the 450 nm region and the high one in the 310 nm range indicates the presence of enzyme-bound reduced flavin (spectra recorded after 2h at RT in an oxygen atmosphere). Trace b, spectra of the same sample upon heat-denaturation of the protein (5 min 100°C). Trace c; spectrum of PDS upon addition of 200 μM DPQ (recorded after 20 min). B, Dependence of the rate of ζ-carotene formation on the Eo’and structure of the quinone electron acceptor. The structures of the naphtoquinones (☐) and benzoquinones (○) used are given in S3 File. Decyl-plastoquinone (100 mV) is highlighted (●).
Fig 9.
Effect of the redox state of FAD and of norfluorazon on the stability of PDS-His6.
Thermo-FAD measurements were carried out in a RealTime-PCR instrument using the SYBR-Green Channel (Exc = 488 nm; Em = 520 nm). Trace 1; PDS-His6 isolated in the absence of norflurazon containing FADox. Trace 2; PDS-His6 isolated in the absence of norflurazon containing FADox after addition of 50 μM norflurazon, Trace 3; PDS isolated in the presence of 50 μM norflurazon containing FADred.