Fig. 3: Structural and mutagenesis analysis of Apg.
From: Molecular insights of acarbose metabolization catalyzed by acarbose-preferred glucosidase
Simulated annealing omit maps are shown in gray mesh and contoured at 1.0 σ. Hydrogen bonding interactions are indicated with gray dashed lines. A Clip diagram of the substrate acarbose in the lumen of the active site of Apg. The hydrophobic surface in blue and the hydrophilic surface in white. Acarbose is indicated by cyan color of stick. The 2Fo-Fc electron density maps of acarbose are shown as gray grids with contours at the 1.0 σ level. B Crystal structure of Apg(D448A)-acarbose (PDB 9IVZ). The substrate acarbose is shown in cyan, residues, in pink, hydrogen bonding interactions in gray dashed lines, and water molecules in red spheres. Validation of the electron density of acarbose-bound and active-ligand water is shown in Supplementary Fig. 7. C Apg(D336A) crystal structure of M1 (PDB 9IZE) (ligand, palegreen; residues, pink). D Crystal structure of Apg(D336A)-M2 (PDB 9IZO). Product M2 is shown in slate, residues, in pink. Stereo views of B-D see Supplementary Fig. 8. E Apg(D448A)-acarbose complex (PDB 9IVZ) (colored as in A) superimposed on mutant Apg(D448A) (PDB 9IXH) (residues, white). Significant conformational changes are observed for W292, Y295. The direct loop domains of Y295 and W292 are shifted to a certain extent. Among them, Y295 has a shift distance of 2.6 Å (atom OH) and W292 has a shift distance of 5.1 Å (atom NH). Maximum displacement of capping loop B to the sugar portion is 4.1 Å (defined by the NH2 atom of D291). F Mutagenesis experiment of residues in the active site of Apg. Asterisks indicate mutants with no measurable activity. Data are presented as mean values ± SD, n = 3. Error bars represent the standard deviation from three repeats.
