Volume 9, Issue 2, Pages 195-204 (February 2016) Structural Analyses of Short-Chain Prenyltransferases Identify an Evolutionarily Conserved GFPPS Clade in Brassicaceae Plants  Chengyuan Wang, Qingwen Chen, Dongjie Fan, Jianxu Li, Guodong Wang, Peng Zhang  Molecular Plant  Volume 9, Issue 2, Pages 195-204 (February 2016) DOI: 10.1016/j.molp.2015.10.010 Copyright © 2016 The Author Terms and Conditions

Figure 1 In Vivo and In Vitro Assays of Arabidopsis GGPPSL Proteins 1–4, 6–11. (A) Schematic diagram for the carotenoid (zeaxanthin β-diglucoside) biosynthesis pathway used for GGPPS complementation in E. coli. (B) Carotenoid production of E. coli harboring the pACCAR25ΔcrtE plasmid and the pET32a vector carrying Arabidopsis GGPPSL genes. The hops large subunit of heteromeric G(G)PPS (GenBank: FJ455407) and empty pET32a vector were used as a positive control (P.C.) and a negative control (N.C.), respectively. (C) Relative quantification of carotenoids produced in E. coli harboring the pACCAR25ΔcrtE plasmid and the pET32a vector carrying Arabidopsis GGPPS homolog genes. Error bars indicate the SE of three replicates. Absence of bar indicates not detected. (D) In vitro enzymatic assay. The purified recombinant GGPPSL proteins (1 μg) were assayed using 14C-IPP and allylic substrate (DMAPP, GPP, FPP, or GGPP) at 30°C for 30 min. The reaction products were separated by TLC. GOH, geranol; FOH, farnesol; GGOH, geranylgeranol; GFOH, geranylfarnesol; PPOH, polyprenol (≥C30). Molecular Plant 2016 9, 195-204DOI: (10.1016/j.molp.2015.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 2 Structures of AtGFPPS2, AtPPPS2, and AtGGPPS11. (A) Overall structure of an AtGFPPS2 monomer (top view). The structural cartoon is colored in rainbow, from blue (N terminus) to red (C terminus). The FARM and SARM motifs, the I site, and the product elongation pocket are indicated. (B) Superimposition of AtGFPPS2, AtPPPS2, and AtGGPPS11 structures show the difference. Structures are shown with ribbons, and are colored in light blue (AtGFPPS2), green (AtPPPS2), and magenta (AtGGPPS11). (C–F) The product elongation pockets of AtGGPPS11 (C), AtGFPPS2 (D), AtPPPS2 (E), and AtGGPPS1 (F). Helices (D–F) are shown in light blue. Side chains of the residues forming the pocket “floors” of AtGGPPS11 (Met80, Phe123), AtGFPPS2 (Phe145), and AtGGPPS1 (Arg132) are colored orange. The structure of AtGGPPS1 was modeled using SWISS-MODEL). Molecular Plant 2016 9, 195-204DOI: (10.1016/j.molp.2015.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 3 The “Three Floors” Model and Mutational Analyses. (A) The “three floors” model is illustrated using AtGFPPS2. For clarity, a substrate FPP (magenta sticks) is modeled into the structure using 1UBX. The side chains of the residues forming the “floors” are shown with sticks in different colors, from top to bottom: green (floor 1, residues Ser102, Ser103, and Leu177), gold (floor 2, residues Leu141 and Ile172), and orange (floor 3, residues Phe145 and Leu168). (B) Results of in vitro activity of AtGGPPS11 and mutants. Purified wild-type and mutated proteins were assayed using 14C-IPP and DMAPP as co-substrates, and the reaction products were separated by TLC. (C–E) Results of in vitro assay of wild-type and mutated AtGFPPS2 (C), AtPPPS2 (D), and AtGGPPS11 (E). DMAPP and 14C-IPP were used as co-substrates in all reactions, and the products were analyzed using TLC. GOH, geranol; FOH, farnesol; GGOH, geranylgeranol; GFOH, geranylfarnesol; PPOH, polyprenol (≥C30). Molecular Plant 2016 9, 195-204DOI: (10.1016/j.molp.2015.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 4 Identification of GFPPS and PPPS in Brassicaceae Plants. (A) GFPPS and PPPS from Brassicaceae plants were predicted based on the “three floors” model. The residues constituting the product elongation pocket helices and three floors are aligned. Invariant residues are highlighted in red, and conserved amino acids are boxed. The secondary structural elements of AtGFPPS2 are shown above the aligned sequences. The floor-1, -2, and -3 residues are indicated by green triangles, golden squares, and orange stars, respectively. Aly, Arabidopsis lyrata; Cru, Capsella rubella; Bst, Boechera stricta; Bra, Brassica rapa; Esa, Eutrema salsugineum. (B) In vitro activity of predicted GFPPS and PPPS. All of the predicted GFPPS and PPPS enzymes were characterized using DMAPP and 14C-IPP as co-substrates; the products were analyzed by TLC. AtGFPPS2, AtPPPS2, and AtGGPPS11 were used as positive controls. Molecular Plant 2016 9, 195-204DOI: (10.1016/j.molp.2015.10.010) Copyright © 2016 The Author Terms and Conditions

Figure 5 Phylogenetic Analysis of GGPPSL Proteins and the GFPPS–TPS Gene Clusters Found in Brassicaceae Plants. (A) Phylogenetic analysis of plant GGPPSL proteins, including GGPPS, GFPPS, and PPPS. Bootstrap values (based on 1000 replications) >70% are shown for corresponding nodes. The experimentally confirmed GFPPS and PPPS proteins are highlighted by blue brackets and red spots, respectively. The scale measures evolutionary distance in substitutions per site. Aco, Aquilegia coerulea Goldsmith; Aly, Arabidopsis lyrata; Ag, Abies grandis; At, Arabidopsis thaliana; Atr, Amborella trichopoda; Cgr, Capsella grandiflora; Cpa, Carica papaya; Cre, Chlamydomonas reinhardtii; Cru, Capsella rubella; Bst, Boechera stricta; Bra, Brassica rapa; Esa, Eutrema salsugineum; Os, Oryza sativa; Pa, Picea abies; Ppa, Physcomitrella patens; Smo, Selaginella moellendorffii; Tha, Tarenaya hassleriana. (B) GFPPS–TPS gene clusters found in the published genomes of Brassicaceae plants. Molecular Plant 2016 9, 195-204DOI: (10.1016/j.molp.2015.10.010) Copyright © 2016 The Author Terms and Conditions