Volume 9, Issue 5, Pages 347-353 (May 2001) Analysis of the Structure, Substrate Specificity, and Mechanism of Squash Glycerol-3- Phosphate (1)-Acyltransferase  Andrew P. Turnbull, John B. Rafferty, Svetlana E. Sedelnikova, Antoni R. Slabas, Ted P. Schierer, Johan T.M. Kroon, J.William Simon, Tony Fawcett, Ikuo Nishida, Norio Murata, David W. Rice  Structure  Volume 9, Issue 5, Pages 347-353 (May 2001) DOI: 10.1016/S0969-2126(01)00595-0

Figure 1 Structure-Based Sequence Alignment for Members of the G3PAT Family The secondary structure assignment for squash G3PAT is represented by cylinders (α helix) and arrows (β strand) above the aligned sequences [3–5, 12–14]. The down arrow (∇) above the sequence alignment represents the previously reported squash G3PAT processing site [4] that was lately found to have been truncated during the purification of the mature enzyme. A filled down arrow (▾) above the aligned sequences represents the current putative processing site [1]. Fully conserved residues are highlighted in reverse type, whereas residues that are fully conserved in chilling-resistant plants (arabidopsis, pea, and spinach) but differ in chilling-sensitive plants (squash and cucumber) are highlighted in gray. An asterisk or plus sign below the sequence alignment denotes a residue that is implicated as being important in binding the fatty acyl substrate or glycerol 3-phosphate, respectively. The figure was prepared using ALSCRIPT [25] Structure 2001 9, 347-353DOI: (10.1016/S0969-2126(01)00595-0)

Figure 2 Stereo Diagrams of Squash G3PAT (a) A schematic representation with the strands and helices labeled and colored red and green, respectively. The figure was prepared using MOLSCRIPT [26]. (b) Cα trace with every tenth residue dotted and every twentieth residue numbered. The pattern of sequence conservation across members of the G3PAT family is also illustrated. Residues that are fully conserved across all five representative G3PAT sequences are colored green, residues that are fully conserved in chilling-resistant (arabidopsis, pea, and spinach) plants but differ in chilling-sensitive plants (squash and cucumber) are highlighted in red, and the remainder are highlighted in blue. The modeled positions of the glycerol 3-phosphate and fatty acyl substrate moieties are shown in atom colors (carbon, white; oxygen, red; and phosphate, pink) and cyan, respectively. The figure was prepared using MIDAS PLUS [27, 28] Structure 2001 9, 347-353DOI: (10.1016/S0969-2126(01)00595-0)

Figure 3 Stereo Diagrams of the Proposed Substrate Binding Sites in Squash G3PAT The glycerol 3-phosphate moiety is displayed in atom colors (carbon, white; oxygen, red; and phosphate, pink), with the fatty acyl substrate and pantotheine linker colored cyan. Residues are colored according to the criterion outlined in Figure 2b. (a). The presumptive glycerol 3-phosphate binding site. Residues with at least one atom residing within 10 Å of the modeled glycerol 3-phosphate position are shown. The phosphate moiety of glycerol 3-phosphate lies in a positively charged pocket formed by the side chains of two arginines (residues 235 and 237), a lysine (193), and a histidine residue (194), and the substrate's C1-hydroxyl group is positioned in the same orientation as the analogous hydroxyl group of the catalytic serine in the serine proteases, with His-139 and Asp-144 (which form the conserved H(X)4D motif) completing the catalytic triad. The superimposed catalytic triad for the serine proteases is highlighted in cyan. (b). The proposed fatty acyl substrate binding site in squash G3PAT. Several alternative conformations of the fatty acyl side chain are presented in order to illustrate the potential uncertainty in the modeling. The 12 residues residing within 5 Å of the first 9 carbon atoms of the fatty acyl chain are labeled in black, whereas the 14 residues with at least one atom within 10 Å of the average position of the modeled terminal carbon atom of the fatty acyl chain are highlighted in red. Additionally, the residues that are implicated as being important in the binding of the glycerol 3-phosphate are enclosed in brackets. The figure was prepared using MIDAS PLUS [27, 28] Structure 2001 9, 347-353DOI: (10.1016/S0969-2126(01)00595-0)

Figure 4 Stereo Diagram of the Electrostatic Surface of Squash G3PAT Highlighting the Deep Tunnel That Forms Part of the Surface of Domain II Red corresponds to a surface potential of less than −20 kcal/[mol·electron], and blue corresponds to a potential greater than +20 kcal/[mol·electron]. The characteristics of this surface strongly suggest that this region represents the fatty acyl substrate binding site. The modeled positions of the fatty acyl substrate and pantotheine linker are colored cyan. The figure was produced using GRASP [29] Structure 2001 9, 347-353DOI: (10.1016/S0969-2126(01)00595-0)

Figure 5 Stereo Diagram Illustrating the Electrostatic Surface Potential of a Second Deep Pocket Bounded by Hydrophobic Residues That Lie on the Opposite Side of the H(X)4D Site to That Occupied by the Proposed Fatty Acyl Chain Binding Pocket Red corresponds to a surface potential of less than −20 kcal/[mol·electron], and blue corresponds to a potential greater than +20 kcal/[mol·electron]. The modeled position of the pantotheine linker of the fatty acyl substrate is colored cyan, and the position of the N terminus of squash G3PAT is labeled. The shape of this pocket is not consistent with it acting as the binding site for the fatty acyl chain and may represent the region that accommodates additional N-terminal residues that are not present in the G3PAT construct whose structure is described in this paper. The figure was produced using GRASP [29] Structure 2001 9, 347-353DOI: (10.1016/S0969-2126(01)00595-0)