Fig. 4: Active site, crystallographic complexes, and XU^m4X-recognition.

a Aldouronic acid ligands in the complexes dΔS270A:U^m4X, dΔS270A:U^m4XX^-OH, and dΔS270A:XU^m4XX^-OH. 2F_o–F_c electron density maps for the refined ligands are shown at the 1.0 σ level. b Apo-Δ*dWT with an intact active site (pH 5.5). Hydrogen bonds between residues in the catalytic triad are indicated. c Apo-dΔS270A (pH ca. 7.5) with Y366 in a different conformation (molecule B only). Loss of interactions to S270 might explain the slight destabilization observed for the inactive variant (Fig. 1d). d Ligand-bound structure, dΔS270A:U^m4X(X) (pH ca. 7.5), shown in the same orientation as in b, c for a direct comparison with the unliganded structures. Note that two movements are observed upon substrate binding: Y366 moves “out”, whereas R271 moves “in” and participates in substrate recognition and formation of the oxyanion hole. e dΔS270A:U^m4X(X) complex shown in a different perspective to visualize the extensive network of hydrogen bonds in recognition of the 4-O-methyl-α-d-glucuronoyl moiety. f, g Two different views on aldouronic acid ligand recognition mode focusing on interactions with the xylo-oligosaccharide backbone. The superposition of the dΔS270A complexes with U^m4X, U^m4XX^-OH, and XU^m4XX^-OH, based on the protein C_α atoms, illustrates little variation in the position of the ligand. Residues within 5 Å from the xylosyl moieties are labeled and shown in black. h dΔS270A:U^m4XX^-OH, aldotetrauronic acid in complex with the inactive variant. i Δ*dWT:U^m4XX^-OH, aldotetrauronic acid in complex with catalytically active CuGE. Continuous electron density between the catalytic nucleophile S270 and the ligand suggests the formation of a covalent reaction intermediate.