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Overview
Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encoding the lysosomal enzyme, acid-β-glucosidase (glucocerebrosidase, GlcCerase, E.C. 3.2.1.45 ). The most common treatment for Gaucher disease is enzyme replacement therapy (ERT), in which defective GlcCerase is supplemented with an active enzyme.
Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the approximately 200 GlcCerase mutations and disease severity, although homozygosity for the common is associated with non- neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 A resolution. The catalytic domain consists of a (beta/alpha)(8) TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the is consistent with a catalytic mechanism of retention. N370 is located on the longest alpha-helix (), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the and a separate on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.
The crystal structure of the human acid-β-glucosidase (colored yellow) with covalently bound irreversible inhibitor (conduritol-B-epoxide; CBE; shown in cyan with its hydroxyl groups are in red) was solved. This structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the active-site catalytic nucleophile, because the between the cyclohexitol C1 atom and Glu340 Oε2 is 1.43 Å. The comparison between the active sites of and another representative of the glycohydrolase family - plant , reveals that CBE bound with this plant enzyme adopted the "chair" conformation, while with human , it is observed in a "boat" conformation, with hydrogen bonds to Asn234 Oδ1 and Nδ2, Glu340 Oε1, Trp179 Nε1, and Asp127 Oδ1 and Oδ2.
Only one of two of a pair of flexible loops (L1: Ser345–Glu349, and L2: Val394–Asp399) located at the entrance to the active site in native GlcCerase (1ogs) is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE (colored blue), while these loops in the second (closed) conformation are colored magenta. In , a major structural change is observed in the positions of , and in a more limited difference is observed in the conformations of . Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. The movies 1 and 2 illustrate the dynamics of the movement of these two loops (Refs 1,2).
