Beta-glucosidase

From Proteopedia

Revision as of 15:23, 8 November 2009

Template:STRUCTURE 2vrj

Introduction

Origin and structure

2VRJ is a β-glucosidase which EC number is 3.2.1.21. 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. β-glucosidase is in complex with N-octyl-5-deoxy66-oxa-N-carbamoylcalystegine. In terms of structure 2VRJ is a homodimer. It means that it is composed of two chains and which are chiral. 438 residues are involved in each chain and constitutes a subunit of the protein. Each subunit contains a catalytic site. β-glucosidase is called β-D-glucoside glucohydrolase or cellobiase too.

Biocatalyst

A β-glucosidase is an enzyme which catalyses the hydrolysis of terminal non-reducing residues in β-glucosides. 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.

2VRJ

Structure and function

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. β-glucosidases are exocellulases. It means that they act only on the end of the molecule they have to hydrolyse. It implies that the catalytic site has to be near the surface of the protein so that the residues which compose the catalytic site have to be hydrophilic. Glutamate and asparagin are hydrophilic amino-acids . 2VRJ presents two catalytic sites composed of three residues : two residues of glutamate()and one residue of asparagin (). The protein is presented in complex with an inhibitor called .We can see that the two glutamate residues involved in the catalytic site are really closed to the ligand. Indeed there are interactions between these residues and calystegine.

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. 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 10.5 amgstroms.

Retaining glycoside hydrolases

Retaining glycoside hydrolases 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 permits the deglycosylation of the enzyme. In this case, there are two transition states involved. The distance between the two carboxylates is about .

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 stabylise 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. Cellulase, the enzyme can better access the structure to act on it. The second step: hydrolysis is the most important. 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.

References

http://www.cazy.org/fam/ghf_INV_RET.html#3 http://www.ebi.ac.uk/pdbe-srv/view/entry/2vrj/viewer http://www3.interscience.wiley.com/cgi-bin/fulltext/121428480/HTMLSTART