The 1.9 Å Structure of α-N-Acetylgalactosaminidase Scott C Garman, Linda Hannick, Alex Zhu, David N Garboczi  Structure  Volume 10, Issue 3, Pages 425-434 (March 2002) DOI: 10.1016/S0969-2126(02)00726-8

Figure 1 Overall α-NAGAL Reaction and Structure (A) The α-NAGAL reaction. α-NAGAL hydrolyzes the α1→R linkage of a substrate containing a terminal α-NAGAL sugar, releasing the R substituent and free α-GalNAc. Both the substrate and product are α anomers, as they contain axial substituents at C1. (B) The α-NAGAL dimer in ribbon representation. Domain 1 (blue) bears the active site containing the reaction product α-GalNAc (yellow). Domain 2 (red) packs against domain 1. Structure 2002 10, 425-434DOI: (10.1016/S0969-2126(02)00726-8)

Figure 2 The α-NAGAL Structure All four panels show the structure with a color gradient along the amino acid sequence, from the N terminus (blue) to the C terminus (red). (A) A topology diagram shows the secondary structure of the monomer. Domain 1 (on the left) contains alternating β strands and α helices, while domain 2 (on the right) contains an antiparallel β sheet. (B and C) Ribbon diagrams of the polypeptide fold, as seen from the top and rotated 90°. Domain 1 is on the left in these two panels. The α-GalNAc ligand is shown as bonds colored by atom type. (D) A stereo diagram of the α-carbon trace of the α-NAGAL dimer. The ligand is shown as in (B) and (C). Structure 2002 10, 425-434DOI: (10.1016/S0969-2126(02)00726-8)

Figure 3 The Active Site of α-NAGAL (A) The 1.9 Å electron density and active site residues from the native data set. A glycerol molecule appears in the active site, mimicking the location of the ligand. (B) The 2.4 Å electron density, α-GalNAc sugar, and active site residues from the protein complex with ligand, with residue numbers indicated. The orientation is identical to (A), and residues are colored as in Figure 2. The gray electron density maps (σa-weighted 2Fo−Fc-simulated annealing composite omit) are contoured at 1.6 σ. (C) A cartoon diagram summarizing the interactions between the protein and α-GalNAc. The ligand is shown in bold, hydrogen bonds less than 3 Å in length are shown as red dashed lines, longer hydrogen bonds are shown as gray dashed lines, and van der Waals contacts are shown as blue dotted lines. Yellow boxes highlight residues that disrupt catalytic activity when mutated. Structure 2002 10, 425-434DOI: (10.1016/S0969-2126(02)00726-8)

Figure 4 Active Site and Catalytic Mechanism (A) Two views of the α-NAGAL molecular surface. At left, an acidic patch (in red) connects the two active sites of the dimer, with the ligand depicted in yellow. The view at right is rotated 180°. The electrostatic potential on the molecular surface is plotted from −10 kT (red) to +10 kT (blue), as calculated by the program GRASP [43]. (B) The two-step catalytic mechanism is shown in a cartoon. Step 1: Asp140 from α-NAGAL makes a nucleophilic attack on C1 of the terminal α-N-acetylgalactosamine of the substrate, cleaving the glycosidic linkage and producing a covalent intermediate. Step 2: a water molecule, deprotonated by Asp201, makes a nucleophilic attack on C1 of the substrate, cleaving the covalent enzyme-substrate intermediate complex. This two-step mechanism results in a product with the same α-anomeric state as the starting substrate. (C) Conserved active sites in glycosidases. Two α-retaining glycosidases are superimposed by their bound ligands. Each enzyme contains carboxylic acids arranged on opposite sides of the bond to be cleaved. α-NAGAL is in red and α-amylase (PDB code: 1JIB) is in blue. The labile glycosidic linkage is marked with a large atom. Dotted lines connect the appropriate carboxylate oxygens to the ligand. Some ligand atoms have been truncated for clarity. (D) Convergent evolution of glycosidases. Four enzymes of distinct fold families but related function are superimposed by their bound ligands. Each of the enzymes contains two carboxylic acids arranged on opposite sides of the bond to be cleaved. α-NAGAL is in red, glucoamylase (PDB code: 1GAH) in yellow, lysozyme (1LZC) in green, and xylanase (1B3X) in gray, and the objects are drawn as in (C). Structure 2002 10, 425-434DOI: (10.1016/S0969-2126(02)00726-8)

Figure 5 Sequence Alignment of Homologous Enzymes Sequence alignment of chicken α-NAGAL (Ch α-NAGAL), human α-NAGAL (Hu α-NAGAL), and human α-GAL (Hu α-GAL). Sequence identities are colored yellow, and conservative substitutions are colored green. Overall, there is a high degree of amino acid sequence identity among the related enzymes: chicken α-NAGAL and human α-NAGAL have 75% identity; chicken α-NAGAL and human α-GAL have 54% identity; and human α-NAGAL and human α-GAL have 50% identity. Some residues affecting catalytic activity, Trp16, Asp61, Tyr103, and Asp140, are marked with red arrows. Ser172 and Ala175, on the N-acetyl recognition loop, are shown with black arrows. Note the replacement of Glu for Ser and Leu for Ala in the α-GAL sequence. Structure 2002 10, 425-434DOI: (10.1016/S0969-2126(02)00726-8)

Figure 6 Human α-NAGAL and α-GAL Models Showing the Extent of Sequence Similarity and the Location of Disease Mutations (A) Model of human α-NAGAL (top left, with 75% identity) colored by sequence similarity to chicken α-NAGAL. (B) Model of human α-GAL (lower left, with 54% identity) colored as in (A). Note the large number of identical and conserved residues (yellow and green), particularly around the active sites, shown with bound ligand (red). Residues that are not conserved (blue) are mainly on the surface and in loops. (C) The human α-NAGAL model (top right) showing the locations of Schindler and Kanzaki disease-causing mutations. (D) The human α-GAL model (bottom right) displaying 105 locations where Fabry disease-causing point mutations have been identified. The mutations are distributed throughout the structure. Mutations leading to mild or severe forms of disease are colored orange or cyan, respectively. Each model is shown as an α-carbon trace. Structure 2002 10, 425-434DOI: (10.1016/S0969-2126(02)00726-8)