Fig. 5. Structure analysis of 8-mer 1 Binding to 3-OST-3 (A) Position of the two 8-mer 1 oligosaccharides in the active site of 3-OST-3 based on the superposition of the two 3-OST-3 molecules (molecule B of 3-OST-3 shown, RMSD of 0.40 Å over 257 Cα atoms). 8-mer-1 (green) from active site of molecule B (gray). The 8-mer 1 from molecule A is colored all in light blue. Saccharides are labeled as in Fig. 1B. (B) Binding interactions of 3-OST-3 molecule B with 8-mer 1 substrate (green) and PAP cofactor product (cyan). Potential hydrogen bonds are shown in black dashed lines, and the bound sodium ion is colored purple with a nearby iodide ion from the crystallization condition colored aqua. A red asterisk denotes the position of the acceptor 3-OH for the sulfo transfer. (C) Superposition of the structure of 3-OST-1 (pink, PDB ID code 3UAN²¹ molecule A) onto 3-OST-3 molecule B bound to 8-mer 1 (RMSD of 0.87 Å over 248 Cα atoms). Conserved residues in 3-OST-1 and -3 previously shown to be important for activity (Lys162, Arg166, Glu184, His186, Asp189, Lys215, Gln255, Lys368, Arg370) are displayed for both enzymes. Shown in magenta are the two gate-residues of 3-OST-1, His278 and Glu88 (sidechain is disordered and not modeled), that are important for 3-OST-1 specificity.¹⁷ The hydrogen bond between the catalytic base Glu184 and acceptor 3-OH is represented with a dashed line, while the trajectory of the in-line transfer from the acceptor 3OH to the leaving group PAP is highlighted with a red dashed line. (D) Chemical structures of the ordered saccharides from the 8-mer 1 substrate bound to 3-OST-3 (top) and the chemical structure of the visible saccharides from the 7-mer bound in the 3-OST-1 structure (3UAN²¹) (bottom).