Beta-glucosidase
β-glucosidase is an enzyme which catalyses the hydrolysis of terminal non-reducing residues in β-glucosides (EC number : 3.2.1.21). In the case of 2VRJ, it comes from Thermotoga maritima which is a rod-shaped bacterium belonging to the order of Thermotogates. This bacterium was originally isolated from geothermal heated marine sediments. 2VRJ is here is in complex with an inhibitor called N-octyl-5-deoxy66-oxa-N-carbamoylcalystegine [1].
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ContentsGeneral action as biocatalystIt acts on the β(1-4) bond linking two glucose residues or glucose-substituted molecules. The action of the enzyme on such glucosides results in the release of units of glucose. For instance, hydrolysis of cellobiose catalysed by a β-glucosidase releases two glucoses [4].   β-glucosidases can also be called β-D-glucoside glucohydrolases or cellobiases. 2VRJStructure and functionIn terms of structure 2VRJ is a homodimer. It means that it is composed of two chains A and B which are chiral. Each chain is composed of 438 residues and constitutes a subunit of the protein. Each subunit contains a catalytic site. The enzymatic hydrolysis of a glycosidic bond requires two critical residues : a proton donor and a proton acceptor which can also be called a nucleophile/base. Aspartate and glutamate have been found to perform catalysis [5]. Accorded to this, studies showed that one of the conserved regions of β-glucosidases is centred on conserved glutamate residues [6]. As every β-glucosidase, 2VRJ presents two conserved residues of glutamate (166 and 351). Moreover 2VRJ has a third important residue : asparagin 293 [7]. The protein is presented in complex with an inhibitor called calystegine. We can see that the two glutamate residues and the asparagin are really closed to each other and to the ligand. Such a proximity highly suggests that there are important interactions between them. So we can say that the catalytic site of 2VRJ is composed of two glutamate and one asparagin. There are three different topologies for the active site of β-glucosidases : the pocket or crater, the cleft or groove and the tunnel [8]. The topology of 2VRJ active site is a pocket in which the ligand can bind. Hydrolysis of terminal non-reducing residues in β-glucosidesThere are two ways to hydrolyse the terminal non-reducing residues in β-glucosides which implicate the two glutamate residues and a molecule of water [9]. Water which is an amphoter, is here used as a base for the nucleophilic attack on the positively charged anomeric carbon. The general equation of the chemical reaction is :  Inverting glycoside hydrolasesInverting glycoside hydrolases lead to an inversion of the anomeric configuration to create an alpha configuration. The steps of the reaction are like the mechanism of nucleophilic substitution S2N. It is an one step process: the nucleophile (water) the anomeric carbon with simultaneous expulsion of the leaving group (OR). Bond making takes place at the same time as bond breaking. Such a mechanism is called concerted reaction. The distance between the two carboxylates is about 10.5 angströms. Retaining glycoside hydrolasesRetaining glycoside hydrolyses occur in two steps: the first step, called glycosylation leads to the release of the leaving group and the creation of a carbocation. Subsequently, water attacks this last one. The second step, called deglycosylation consists of OR- nucleophilic attack on the intermediate and allows the deglycosylation of the enzyme. In this case, there are two transition states involved. The distance between the two carboxylates for this mechanism is about 5.5 angströms. For 2VRJ the distance between its two glutamates is about 5 angströms. So we can say that 2VRJ seems to be a retaining enzyme. NB: The values of the pH and the nature of the solvent play a main role in the rate of the reaction. Glutamates are directly involved in the catalytic reaction but asparagine is used to stabilise the structure. Other use of β-glucosidasesβ-glucosidase is now used for the synthesis of biofuel. Wood is an abundant and renewable energy which can be changed into bioethanol thanks to enzymatic hydrolysis. This synthesis needs five steps. First it is pre-hydrolysis. The structure is divided into lignin and (hemi)cellulose. The cellulase can better access the structure to act on it. The second step: hydrolysis is the most important. A cellulase is a complex of 3 enzymes which act together to hydrolyse cellulose: Endoglucanase breaks the chain in the middle of the molecular structure of cellulose. Exoglucanase binds an available end of the chain and isolates it. Then units of cellobiose are cut (two units of glucose which are together). To finish, β-glucosidase divides cellobiose into two glucoses. When they ferment, they become ethanol. The final product is obtained thanks to fermentation, distillation and deshydratation. Treatment of Gaucher diseaseGaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encoding the lysosomal enzyme, acid-β-glucosidase (acid-beta-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. The correlation between the ~ 200 mutations in GlcCerase and disease severity is not completely understood, although homozygosity for the common mutations N370S and L444P is associated with non-neuronopathic and neuronopathic disease, respectively. Imiglucerase (Cerezyme®)The X-ray structure of GlcCerase (Cerezyme®) was resolved at 2.0 A resolution (1ogs). The catalytic domain consists of a (beta/alpha)(8) TIM barrel, as expected for a member of the glucosidase hydrolase A family. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest alpha-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain 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 [10]. GlcCerase with cyclohexitolThe crystal structure of the human colored yellow with covalently bound irreversible inhibitor cyclohexitol (conduritol-B-epoxide; CBE; shown in cyan with its hydroxyl groups are in red) was solved (1y7v, [11]). 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 distance between the cyclohexitol C1 atom and Glu340 Oε2 is 1.43 Å. The comparison between the active sites of GlcCerase and another representative of the glycohydrolase family - plant β-D-glucan glucohydrolase (1iev, [12]), reveals that CBE bound with this plant enzyme adopted the "chair" conformation, while with human GlcCerase, it is observed in a "boat" conformation, with hydrogen bonds to Asn234 Oδ1 and Nδ2, Glu340 Oε1, Trp179 Nε1, and Asp127 Oδ1 and Oδ2 [11]. Only one of two alternative conformations 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 (1y7v), 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 loop 2, a major structural change is observed in the positions of Asn396 and Phe397, and in loop 1 a more limited difference is observed in the conformations of Lys346 and Glu349. 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 [11][13]. Native human acid β-glucosidase, expressed in cultured plant cells (prGCD, pGlcCerase)Three-dimensional structure of recombinant plant-derived glucocerebrosidase (prGCD, 2v3f) consists of 3 domains. Domain I (residues 1–27 and 384–414, colored pink) comprises a 3-stranded anti-parallel β-sheet flanked by a perpendicular amino-terminal strand. Domain II (residues 30–75 and 431–497, colored lime) consists of two β-sheets. Domain III (residues 76–381 and 416–430, colored red) is a (β/α) 8 TIM barrel. The catalytic site with molecule BTB is shown [14]. Structural alignment of prGCD (2v3f) with both Cerezyme® (1ogs) and Cerezyme® covalently modified by an irreversible inhibitor, conduritol-B-epoxide, colored yellow (1y7v), revealed highly significant structural identity. The RMSD values for Cα atoms of these structures were of 0.64 and 0.60 Å, respectively. Moreover, there was strict conservation of the active site residues [14]. pGlcCerase with ligandsSuperimposition of the structure of native human acid β-glucosidase, expressed in cultured plant cells (pGlcCerase, 2v3f) on those of N-butyl-deoxynojirimycin/pGlcCerase (2v3d), N-nonyl-deoxynojirimycin/pGlcCerase (2v3e), and isofagomine/deglycosylated Cerezyme (IFG/DG-Cerezyme, 2nsx) reveals significant structural identity, neither of these ligands causes structural changes upon binding to the enzyme. The imino sugar of N-butyl-deoxynojirimycin (NB-DNJ) forms 7 hydrogen bonds and also makes several hydrophobic interactions with side chains of active site residues (2v3d). The crystal structure of pGlcCerase in complex with N-nonyl-deoxynojirimycin (NN-DNJ) (2v3e) is very similar to that of NB-DNJ/pGlcCerase. The exception is that longer chain of NN-DNJ interacts with 2 additional residues Leu241 (labeled lime) and Leu314 of symmetrically related monomer (not shown). Comparison of the structures of NB-DNJ/pGlcCerase (2v3d) and NN-DNJ/pGlcCerase (2v3e) with that of IFG/DG-Cerezyme (2nsx) shows that the pyranose-like ring forms a same number of hydrogen bonds with the enzyme in all three cases (2v3d, 2v3e, and 2nsx) [14][15][16]. Velaglucerase alfaThe structural alignment of the crystal structure of velaglucerase alfa (colored red) (2wkl) reveals that it is very similar to those of the recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®, colored blueviolet, 2j25) and in transgenic carrot cells (prGCD, 2v3f). Superposition of the two individual molecules in the asymmetric unit of velaglucerase alfa and imiglucerase demonstrates striking similarity between positions of catalytic residues E235 and E340 (colored orange) in all 4 molecules. The position of H311 is also very similar in all 4 molecules, whereas the conformations of 3 other active site residues W312, Y313, and, especially N396 are somewhat different. The active site residues (except E235 and E340) of the two individual molecules in the asymmetric unit of velaglucerase alfa are colored: subunit A (red), subunit B (lime) and of imiglucerase: subunit A (blueviolet), subunit B (magenta). Imiglucerase and pr-GlcCerase contain a histidine at residue 495 (blueviolet), whereas velaglucerase alfa contains arginine (red). Mutations which cause Gaucher disease, R496 and D474 are close to R495 near the N-terminus of GlcCerase. The velaglucerase alfa (its glycans are colored blue) and imiglucerase (its glycans are colored magenta) have different carbohydrate composition [14][17][18]. This difference in glycosylation causes the increased cellular uptake of velaglucerase alfa over imiglucerase and could lead to improvement of treatment of Gaucher disease [18]. Additional ResourcesFor additional information, see: Carbohydrate Metabolism 3D structures of Beta-glucosidaseBeta-glucosidase 3D structures
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Proteopedia Page Contributors and Editors (what is this?)
Alexander Berchansky, Muriel Breteau, Michal Harel, David Canner, Joel L. Sussman
