Figure 1. Sequence alignment of DspB with the catalytic domains of β-hexosaminidases from human β-hexosaminidase B (1O7A), human lyzosomal β-hexosaminidase (1NOW), S. marcescens (1QBB) and S. plicatus (1HP4). There are three regions of high sequence identity with patches of conserved residues along the chain. Note that sequence similarity in the loop regions following the β-strands and α-helices is very sparse. The alignment was constructed using ClustalW. Amino acid numbering indicated on the top is for DspB. Secondary structures are indicated along the top of alignment (α, α-helix; β, β-sheet). Homology in the N-terminal end of the sequence is very high compared to the C-terminal end. This Figure was generated using ESPript.⁵⁸

Figure 2. The topology and comparison with the homologues of DspB. (a) Schematic topogram generated by the program TOPS located at the URL (http://www.tops.leeds.ac.uk/). α-Helices are represented by cylinders and β-strands by arrows. (b) Ribbon diagram for the refined structure of DspB showing the TIM barrel down the barrel axis. (c) A 2F_o−F_c difference density map around the putative active site. The map is contoured at 1.5σ. The refined model of DspB has been superimposed. (d) Superposition of 1HP5 (green) and DspB (magenta) by least-squares fit as derived from the program SPDBV (http://www.expasy.org/spdbv/).

Figure 3. Electrostatic potential surface for DspB generated by SPDBV. Superimposed on this surface is the ribbon diagram for 1HP5 (green) showing the inhibitor NAG-thiazoline (green). Note that the absence of loops observed in the 1HP5 structure (shown in green) make the active site of DspB more open. The active site glycerol and acetate ion in DspB is also shown (blue).

Figure 4. Diagrammatic illustration of the interactions formed by the ligands bound in the active sites of DspB (a) and 1HP5 (b). The hydrogen bond interactions are indicated with a broken line. An arc with radiating lines represents the hydrophobic interactions in the structure. The glycerol is labeled as Gol. (c) Superposition of active site residues at subsite −1 of 1HP5 (magenta) with the corresponding residue from DspB (green). Note that the residues in both the enzymes are almost conserved. Note also the absence of the Trp408 residue in DspB and the difference in the loop conformation and length in DspB.

Figure 5. Comparison of the conformation adopted by β(1,4)-linked disaccharide of N-acetyl-d-glucosamine (from the structure of 1QBB compared to that of an energy-minimized structure of β(1,6)-linked N-acetylglucosamine using the Tripos software SYBYL. One of the two glucosamine residues has been fitted to show the differences in the conformation due to the difference in the linkage. Note that if the fitted unit corresponds to the −1 subsite in DspB and 1HP5, then the +1 subsite residue would require a different conformational space.

Data collection and refinement statistics