Volume 25, Issue 8, Pages 1222-1232.e3 (August 2017) Structure of Phytoene Desaturase Provides Insights into Herbicide Binding and Reaction Mechanisms Involved in Carotene Desaturation  Anton Brausemann, Sandra Gemmecker, Julian Koschmieder, Sandro Ghisla, Peter Beyer, Oliver Einsle  Structure  Volume 25, Issue 8, Pages 1222-1232.e3 (August 2017) DOI: 10.1016/j.str.2017.06.002 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 1222-1232.e3DOI: (10.1016/j.str.2017.06.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Carotene Desaturation in Plants The desaturation and isomerization of phytoene to lycopene occurs in four consecutive steps in plants while bacteria convert the substrate using a single enzyme. The desaturation positions and the trans-to-cis isomerization positions are marked in red. IPP, isopentyl-diphosphate; DMAPP, dimethylallyl-diphosphate; GGPP, geranylgeranyl-diphosphate; GGPPS, geranylgeranyl-diphosphate synthase; PSY, phytoene synthase; PDS, phytoene desaturase; Z-ISO, 15-cis-ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotene cis-trans isomerase; CRTI, bacterial phytoene desaturase. The reoxidation of PQH2 through photosynthetic electron transport and/the plastid terminal oxidase (PTOX) is indicated. CRTI utilizes dioxygen as the electron acceptor. For further explanation, see text. Structure 2017 25, 1222-1232.e3DOI: (10.1016/j.str.2017.06.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Crystal Structure of Phytoene Desaturase of Oryza sativa (A) A monomer is colored from blue at the N terminus to red at the C terminus in cartoon representation with the molecular surface representation in light gray. FAD and norflurazon are shown in a ball and stick representation. The α helices are numbered according to Figure 7E. (B) The structure of PDS (PDB: 5MOG) can be subdivided into three domains. The Rossman fold domain and the hydrophobic channel domain are shown in a molecular surface representation while the HotDog fold-like domain is shown as a cartoon. The hydrophobic channel of PDS is shown in blue. (C) The hydrophobic channel of PDS shown in stereo view (wall eyed). The orientation is as shown in (A). The channel is colored by its electrostatic surface potential contoured from −5 to 5 kbT/e with the surrounding amino acids shown as sticks and FAD in a stick and ball representation. (D) The topography of the channel as calculated with Caver 3.0. A molecule of phytoene is shown with its approximate size. The two bonds that undergo desaturation are marked in red while the trans → cis conversion sites are marked with triangles. Water molecules found in the charged part of the channel are shown as blue circles. (E) Waters molecules found in the charged part of the channel are shown as spheres and numbered as in (D). The coordinating amino acids are shown as stick models, FAD and norflurazon as sphere and ball models. An omit electron density map for the water molecules is shown as a green mesh contoured at 4.5 σ. Structure 2017 25, 1222-1232.e3DOI: (10.1016/j.str.2017.06.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Tetrameric Arrangement of PDS in Crystallo (A) PDS is shown as tetramers with each monomer colored individually. The entrances of the hydrophobic channels point toward the center of the tetramer. (B) Close up of the hydrogen bridges between two monomers. (C) At membrane surfaces, a rotation of each monomer is necessary to position the entrance into the lipid bilayer. Structure 2017 25, 1222-1232.e3DOI: (10.1016/j.str.2017.06.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Electrostatic Potential of PDS Shown in Three Orientations The surface potential was calculated using APBS and is contoured from −5 to 5 kbT/e. Structure 2017 25, 1222-1232.e3DOI: (10.1016/j.str.2017.06.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Binding of the Herbicidal Inhibitor Norflurazon PDS Was Crystallized in a Complex with Norflurazon (A) Norflurazon binding site of O. sativa PDS in stereo view. The protein backbone is shown in cartoon representation with the surrounding amino acids shown as sticks. FAD and norflurazon are shown as a ball and stick model in yellow and teal, respectively. The omit electron density for norflurazon is contoured as a green mesh at a σ level of 3.0 of the Fo − Fc map. The previously described norflurazon resistance mediating amino acids Phe162, Arg300, Val505, and Leu538 are colored pink. (B) Coordination of norflurazon found in the structure of PDS. Hydrophobic interactions are shown as red semi-circles. (C) The structures of PDS inhibitors and its electron acceptor plastoquinone indicate the similarity in binding their modes. Structure 2017 25, 1222-1232.e3DOI: (10.1016/j.str.2017.06.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Putative Binding of Ligands and Proposed Reaction Mechanism of PDS (A) The presumed binding site of plastoquinone shown in the same orientation as in Figure 2A. Amino acids involved in the binding of norflurazon are shown as sticks in blue, and a molecule of plastoquinone is drawn in black. While Arg300 lies in the ideal position for coordination of one carbonyl oxygen of plastoquinone, the carbonyl oxygen of Ala539 might coordinate plastoquinol after its reduction by FADH2. (B) A molecule of phytoene is shown in black in the hydrophobic channel of PDS in its probable position. The dehydrogenation and desaturation sites are colored red. Helices 6 and 7 are omitted for clarity. (C and D) Proposed reaction mechanisms. For explanations, see text. Structure 2017 25, 1222-1232.e3DOI: (10.1016/j.str.2017.06.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Structural Superposition of O. sativa PDS and CRTI from P. ananatis (A) The FAD binding domain of PDS in blue is superimposed upon the corresponding domain of CRTI in white. (B) The hydrophobic channel domain of PDS superimposed with its counterpart domain in CRTI. (C) The active site of PDS. Only polar residues are shown in the vicinity of the FAD co-factor as sticks with amino acids capable of acid-base interactions outlined in cyan. The hydrophobic channel is shown as an outline. (D) The proposed active site of CRTI with all charged residues found in the vicinity of the modeled FAD. (E) Sequence alignment and secondary structure elements (TT, β-turn) of O. sativa PDS and P. ananatis CRTI. Conserved residues are highlighted with a red box while similar residues are colored red. Amino acids Arg148, Asp149, and Arg152 that were proposed to function as bases in the catalytic reaction of CRTI are marked with an asterisk. Residues missing in the structure of CRTI are shaded in gray. Structure 2017 25, 1222-1232.e3DOI: (10.1016/j.str.2017.06.002) Copyright © 2017 Elsevier Ltd Terms and Conditions