Phytoene desaturation – “complex” vs. “simple”.

<p>Left, the plant/cyanobacterial system consisting of the two desaturases, phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS). The pathway involves specific poly-<i>cis</i>-intermediates and results in the formation of 7,9,9′7′-tetra-<i>cis</i>-lycopene ( =  prolycopene). <i>Cis</i>-<i>trans</i> isomerases act at the 9,15,9′-tri-<i>cis</i>-ζ-carotene (Z-ISO) and prolycopene (CRTISO) stage, the latter forming all-<i>trans</i>-lycopene, the substrate for lycopene cyclases. The electron acceptors identified so far for PDS (assumed here to be the same for the related ZDS) are plastoquinone and the plastoquinone:oxygen oxidoreductase PTOX. The necessity for an electron donating branch, resulting in redox chains into which PDS integrates has been suggested. Right, CRTI-mediated phytoene desaturation encompassing all four desaturation steps and one <i>cis</i>-<i>trans</i> isomerization step to form all-<i>trans-</i>lycopene. The desaturase CRTI and the isomerase CRTISO share sequential similarity.</p

Structure of CRTI.

<p>The crystal structure of CRTI (A) is shown in comparison with protoporphyrinogen IX oxidoreductase from <i>Myxococcus xanthus</i> (B; Protein Data Bank 2IVD). Pseudodomains are colored in blue (substrate-binding), orange (non-conserved ‘helical’ or ‘membrane binding) and green (FAD-binding). Image was generated with PyMOL. The non-ordered regions in CRTI are indicated by the numbering of adjacent residues.</p

Effect of FAD on the formation of productive membrane associates.

<p>A, SDS-PAGE of liposome-bound CRTI. Membrane binding was carried out in the presence (+FAD) or absence of FAD (-FAD). CRTI bound to liposomes was analyzed after two washing steps with buffer II (left traces) or after a buffer II and additional high-salt washing step (right traces). B, HPLC analysis of phytoene desaturation catalyzed by membrane-bound CRTI. Trace a; lycopene (2) formation from phytoene (1) by membrane-associated CRTI formed in the presence of FAD but incubated without subsequently adding free FAD (free FAD removed by the washing steps). Trace b, same experiment using CRTI associates prepared in the absence of FAD; the incubation contained 150 µM added FAD. Incubation time was 1 h at 37°C. HPLC trace represents a MaxPlot (250–550 nm).</p

Electron transfer reactions catalyzed by CRTI.

<p>A, Potentiometric measurement of oxygen consumption during phytoene desaturation. B, Phytoene desaturation (lycopene formation) using quinones as electron acceptors. The assays were run under an N₂ atmosphere for 30 minutes otherwise maintaining the standard conditions. The quinones used were menaquinone (−80 mV), phylloquinone (−70 mV), menadione (0 mV), duroquinone (+5 mV), Q10 (+65 mV), naphtoquinone (+70 mV) dichlophenolindophenol (+217 mV) and benzoquinone (+280 mV) all at a concentration of 240 µM. Open squares, naphtoquinones, filled symbols, benzoquinones.</p

LC-MS-MS determination of CRTI-bound cofactors

<p>. Upper Trace, photometric response. Lower trace, Single Reaction Monitoring (SRM) was used to determine the presence of FAD M⁺¹ = 786.2 MS² daughter ions m/z  = 348.1, 439.2 and FMN M⁺¹ = 457.1 MS² daughter ions m/z  = 359.2, 439.1. The respective analyses for NAD(H) and NADP(H) yielded no signals. The separation was carried out using HPLC system 5.</p

Structural alignment of CRTI with five FAD-binding Rossmann fold proteins (Pfam:CL0063) identified by a DALI search.

<p> The proteins are <i>Methanosarcina mazei</i> oxidoreductase (RMSD 4.6; 3 KA7; Seetharaman <i>et al</i>., unpublished), <i>Myxococcus xanthus</i> protoporphyrinogen oxidase (RMSD 4.6; 2 IVD; Corradi <i>et al</i>., 2006), <i>Nicotiana tabacum</i> mitochondrial protoporphyrinogen IX oxidase (RMSD 4.8; ISEZ; Koch <i>et al</i>., 2004), <i>Bacillus subtilis</i> protoporphyrinogen oxidase (RMSD 5.3, 3I6D, Qin <i>et al</i>., 2010) and <i>Rhodococcus opacus</i> L-amino acid oxidase (RMSD 4.8; 2JB2; Faust <i>et </i><i>al</i>., 2007). The secondary structure elements of CRTI have been indicated above the alignment and the colored bar underneath the alignment indicates the domain organisation with the FAD-binding domain (green), the substrate-binding domain (blue), and the non-conserved ‘helical’ or ‘membrane-binding’ domain (orange). Disordered regions in the structure are represented by a dotted line and putative FAD binding residues are indicated by purple circles (hydrophobic interactions) and triangles (hydrophilic interactions). This figure was generated with TEXshade <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039550#pone.0039550-Beitz1" target="_blank">[60]</a>.</p

CRTI switch from desaturase to isomerase activity.

<p>Trace a, showing the elution profile (HPLC system 2) of prolycopene (1) extracted from the liposomes used. Trace b, profile of a 3 h incubation (37°C) of prolycopene in the presence of 30 µg CRTI and 100 µM FAD_red under anaerobic conditions. A tri-<i>cis</i>-lycopene species (2) forms predominantly. Trace c, tri-<i>cis</i>-lycopene was purified and incorporated into liposomes. Trace d, liposomes from c, analyzed after incubation with CRTISO and FAD_red<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039550#pone.0039550-Yu1" target="_blank">[11]</a>. This shows the formation of 7,9-di-<i>cis</i>-lycopene (3), all-<i>trans</i>-lycopene (4) and 5-<i>cis</i>-lycopene (5). Trace e, profile of the iodine-catalyzed isomer equilibrium obtained with the tri-<i>cis</i> species in organic solution. UV/VIS spectra of relevant <i>cis</i>-lycopene species are displayed on the right panels.</p

SDS-PAGE analysis of overexpressed CRTI protein: fractions and purification.

<p>The expected molecular mass of the overexpressed protein is 56 kDa. <i>Lane M</i>, molecular mass markers; <i>lane 1,</i> whole cell lysate after IPTG induction; <i>lane 2</i>, pellet after 12,000 × <i>g</i> centrifugation; <i>lane 3</i>, supernatant after 12,000 × <i>g</i> centrifugation; <i>lane 4,</i> fraction after IMAC purification; <i>lane 5</i>, fraction after GPC-purification.</p

Prolycopene isomerization mediated by CRTI in the presence of ²H₂O.

<p>A, HPLC analysis (HPLC system 4) showing the isomerization of tetra-<i>cis</i>-lycopene (prolycopene, peak 1) into tri-<i>cis</i>-lycopene (peak 2) and 7,9 di-<i>cis</i>-lycopene (peak 3) after a 3 h incubation (37°C) in the presence of 30 µg CRTI and 100 µM FAD_red under anaerobic conditions. B, mass spectra (numbering according to A) of remaining substrate and of products.</p

Substrate binding site and aligning amino acid residues.

<p>The substrate binding site shows the lowest energy conformations of <i>in silico</i>-docked C₁₈ substrate. The isoalloxazine ring of FAD docks into the same site (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039550#pone.0039550.s004" target="_blank">Figure S4</a>). The positions of substrate interacting hydrophobic residues are shown in grey (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039550#pone.0039550.s009" target="_blank">Table S2</a>). See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039550#s3" target="_blank">Discussion</a> for the likely role of the charged amino acids.</p