Beta-glucosidase

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Revision as of 09:51, 15 May 2011

Template:STRUCTURE 2vrj

A 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].

General action as biocatalyst

It 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 ^[2].

β-glucosidases can also be called β-D-glucoside glucohydrolases or cellobiases.

2VRJ

Structure and function

spinqualitylabelsall models Structure of the β-glucosidase 2vrj complexed with N-octyl-5-deoxy66-oxa-N-carbamoylcalystegine (PDB entry 2vrj) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image In terms of structure 2VRJ is a homodimer. It means that it is composed of two chains and 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 [3]. Accorded to this, studies showed that one of the conserved regions of β-glucosidases is centred on conserved glutamate residues [4]. As every β-glucosidase, 2VRJ presents two conserved residues of glutamate (). Moreover 2VRJ has a third important residue : [5]. The protein is presented in complex with an inhibitor called . We can see that the two glutamate residues and the asparagin are really closed to each other and to the . 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 [6]. The topology of 2VRJ active site is a in which the ligand can bind.

Hydrolysis of terminal non-reducing residues in β-glucosides

There are two ways to hydrolyse the terminal non-reducing residues in β-glucosides which implicate the two glutamate residues and a molecule of water ^[7]. 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 hydrolases

Inverting 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 hydrolases

Retaining 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 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-glucosidases

β-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.

Additional Resources

For additional information, see: Carbohydrate Metabolism

3D structures of Beta-glucosidase

3ahx – GBA – Clostridium cellulovorans
3ahy – GB – Trichoderma reesei
3abz – KmGB – Kluyveromyces marxianus
2x40 – TnGB3B – Thermotoga neapolitana
3gno - JrGB residues 38-521 – Japanese rice
2rgl, 2rgm - JrGB residues 29-504
3f4v, 3f5j, 3f5k, 3f5l - JrGB7 (mutant)
3ahz – tGB – termite
3fiy, 3cmj - UbGB catalytic domain (mutant) – Uncultured bacteria
2o9p, 1bga – PpGBB – Paenibacillus polymyxa
2jfe - hGB cystolic – human
2e3z – PcGB – Phanerochaete crysosporium
2dga - wGB residues 1-520 – wheat
2vff – GB – Pyrococcus horikoshii
1oif, 1od0 – TmGB catalytic domain - Thermotoga maritima
1ug6 – GB - Thermus thermophilus
1hxj, 1e1e – ZmGB – Zea mays
1e4l - ZmGB (mutant)
1gon – SsGB – Streptomyces sp.
1qox – GB]] - Bacillus circulans
1tr1 - BpGBB (mutant)]] - Bacillus polymyxa
1cbg – GB cyanogenic – White clover
3aiu - rGB residues 50-568 – rye

Beta-glucosidase complex with sugar

3ai0 – tGB + glucoside
3ac0 - KmGB + glucoside
2x41, 2x42 - TnGB + glucoside
3air – wGB residues 50-569 + glucoside + dinitrophenol
3ais - wGB residues 50-569 (mutant) + glucoside + aglycone
3aiq - wGB residues 50-569 + aglycone
3aiv - rGB residues 50-568 + aglycone
3aiw - rGB residues 50-568 + glucoside + dinitrophenol
3gnp, 3gnr - JrGB residues 38-521 + glucoside
3aht, 3ahv – JrGB7 (mutant) + saccharide
1oin - TmGBA + glucoside
3fiz, 3fj0 - UbGB residues 18-482 (mutant) + glucoside
2zox - hGB cystolic + glucoside
2o9s, 2o9r, 2z1s – PpGBB + saccharide
2o9t - PpGBB + glucoside
1uyq - PpGBB (mutant) + glucoside
1bgg - PpGBB + gluconate
2jie - BpGBB + glucoside
1e4i - BpGBB (mutant) + glucoside
2e40 – PcGB + gluconolactone
1v08 – ZmGB + gluco-tetrazole
1e1f - ZmGB + glucoside
1h49, 1e4n, 1e56 - ZmGB (mutant) + aglycone
1gnx - SsGB + saccharide - Sulfolobus solfataricus

Beta-glucosidase complex with inhibitor

2wbg, 2wc3, 2wc4, 2vrj, 2jal, 2j75, 2j77, 2j78, 2j79, 2j7b, 2j7d, 2j7e, 2j7f, 2j7g, 2j7h, 2j7c, 2ces, 2cet, 2cbu, 2cbv, 1uz1, 1wj3, 1oim – TmGBA + inhibitor
2cer – SsGB + inhibitor
1e55 - ZmGB (mutant) + inhibitor

References

1. ↑ Aguilar M, Gloster TM, Garcia-Moreno MI, Ortiz Mellet C, Davies GJ, Llebaria A, Casas J, Egido-Gabas M, Garcia Fernandez JM. Molecular basis for beta-glucosidase inhibition by ring-modified calystegine analogues. Chembiochem. 2008 Nov 3;9(16):2612-8. PMID:18833549 doi:10.1002/cbic.200800451
2. ↑ http://en.wikipedia.org/wiki/B-glucosidase
3. ↑ Davies G, Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure. 1995 Sep 15;3(9):853-9. PMID:8535779
4. ↑ http://www.ebi.ac.uk/interpro/IEntry?ac=IPR018120#PUB00002205
5. ↑ http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/CSA/CSA_Site_Wrapper.pl?pdb=2vrj
6. ↑ Davies G, Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure. 1995 Sep 15;3(9):853-9. PMID:8535779
7. ↑ http://www.cazy.org/fam/ghf_INV_RET.html#3