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Thermotoga maritima β-fructosidase (invertase)
IntroductionIntroduction
The β-fructosidase (β-D-fructofuranosidase EC 3. 2. 1. 26.) [1] of the thermophilic baterium Thermotoga maritima is an enzyme which hydrolyzes sucrose to release an equimolar mixture of fructose and glucose, called "invert sugar". That is why the other name of the β-fructosidase is invertase.
This enzyme achieves the hydrolysis with a retention molecular mechanism. Actually, the invertase hydrolyses the D-sucrose to form the D-fructose and the D-glucose :
So, the substrate anomeric configuration is conserved in products. The β-fructosidase belongs to the GH32 (Glycoside hydrolysases 32) according to the sequence-based classification of glycoside hydrolysases. The β-fructosidase hydrolyses other substrates like raffinose, inulin, levan or stachyose but here we will focus on sucrose hydrolysis.
Glycoside Hydrolases and GH32 familyGlycoside Hydrolases and GH32 family
Glycoside HydrolasesGlycoside Hydrolases
Glycoside hydrolases (GH) [2] constitute a widespread enzyme group presenting a huge variety of proteins folds and substrate specificities. They are classified into EC 3.2.1 as enzymes catalyzing the hydrolysis of O-, N- and S-linked glycosides. The enzymatic hydrolysis of glycosidic bonds can be realised by two ways : inversion or retention of the product anomeric configuration compared with the substrate anomeric configuration. So we can classify Glycoside hydrolases according to these mechanisms. But we can also make a classification depending on three-dimensional structure or on sequence. The classification based on sequence allowed to define more than 120 families of Glycoside hydrolases : GH9, GH14... The Thermotoga maritima β-fructosidase belongs to the GH32 family.
GH32 FamilyGH32 Family
The GH32 family [3] includes over 370 members from plants, fungi and bacteria. This family contains not only invertases but other fructofuranosidases like insulinase, transfructosidase or inulinase. Family GH32 enzymes are retaining enzymes, so the Thermotoga maritima β-fructosidase is a retaining enzyme, as we said in the introduction.This molecular mechanism appears conserved in the sequences of the different GH32 family members. Members of the GH32 family share a common feature which are two important amino acid residues. These two amino acid residues constitute the catalytic machinery accountable to the glycosidic bond cleavage. And they have been identified as an aspartate situated near the N-terminus and acting as the nucleophile, and a glutamate acting as the general acide/base. The GH32 family enzymes present a particular three-dimensional structure. The core of this structure is built of a five-bladed β-propeller appended to a β-sandwich, consisting of two sheets of six β-strands [4]
Thermotoga maritima β-fructosidase structureThermotoga maritima β-fructosidase structure
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| 1uyp, resolution 1.90Å () | |||||||||
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| Ligands: | , , , | ||||||||
| Activity: | Beta-fructofuranosidase, with EC number 3.2.1.26 | ||||||||
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| Resources: | FirstGlance, OCA, RCSB, PDBsum | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
Global organisationGlobal organisation
The Thermotoga maritima β-fructosidase (invertase) is consists of 432 residues. One molecule of this β-fructosidase is composed, like the other GH32 family enzymes, of two modules[4] :
- a five-bladed β-propeller module, from residu 1 to 295. It is the catalytic module.The β-strands forming the blades in the five β-propeller are stronly twisted, forming an angle of about 90° between the first and the last β-strand of a blade. The five β-sheets of the invertase (I to V), presents a "W" topology. The N-terminal second strands lines the central cavity (where is the catalytic active site) and the C-terminal last strands is a the periphery.
- a β-sandwich module , from residu 306 to 432, in C-terminal region.
These two modules are linked by a ten residue linker.
Before purification, six copies of the invertase are rallied together, as we can see on the 3D representation to the right. The ensemble of six bi-modular molecules are arranged into three dimers and display two-fold symetry each. A dimer is arranged around a pseudo-two-fold axis and brigs the β-sandwich domain of a monomer A in contact with the β-propeller domain of a monomer B and vice-versa.
After purification, the Thermotoga maritima invertase is a in solution and have a size of about 30 kDa. A monomer of this invertase has an elliptical shape. The dimensions of this elliptical monomere are about 63 Å x 43 Å x 45 Å[4]. And there is a negatively charged surface depression at the center of the β-propeller.
The active siteThe active site
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The is located at the end of the cavity previously defined. This active site has a . The three carboxylate groups of two aspartate (Asp17 and Asp138) and one glutamate (Gu190) engender a high negative charge at the active site. The Asp17 tallies with the catalytic nucleophile and the Glu190 is the general acide/base. The two regions, wich contain the catalytic amino acid residues, are highly conserved regions in GH32 family. The crystal structure of the Thermotoga maritima β-fructosidase shows a glycerol molecule, which is in the substrate biding site mimicking the O4 and O6 hydroxyl-groups of the substrate fructose-unit. This structure also shows that the Asp138 forms hydrogen bonds to O3 and O4 of the fructose unit. The Ser75 forms also hydrogen bonds to the O4 hydroxyl of fructose and to the catalytic nucleophile Asp17[4].
| Residues involved in the | Residues involved in the |
| Asp138 : forms hydrogen bounds to O3 and O4 of the fructose unit | Asp17 : catalytic nucleophile |
| Ser75 : forms hydrogen bounds to the 04 hydroxyl of fructose unit | Glu190 : general acide/base |
The β-sandwich moduleThe β-sandwich module
The of the Thermotoga maritima β-fructosidase is made up of amino acid residues 306 to 432. This module is constituted by two sheets of six β-strands and is connected to the five-bladed β-propeller module by a short linker-region (about ten residues) which is wrapped around the β-sandwich[4]. According to sequence alignments, this module have no equivalent in other GH32 family proteins (amino acid residues succesion). This no existing similarity of Thermotoga maritima invertase β-sandwich module, compared to other GH32 family proteins, may be due to the loss of function of this module. Otherwise, it can be due to the evolution of this module to preserve stability at high temperature.
β-fructosidase applicationsβ-fructosidase applications
β-fructosidase has many applications [5] because, as we said before, this enzyme hydrolyzes sucrose to release an equimolar mixture of fructose and glucose ("invert" sugar). This "invert" sugar syrup is very used in food industry because it provides a longer shelf life for products. For instance, invertase prevents cristallization in soft candies like cherry cordials. This enzyme is also used for artificial honey fabrication, pharmaceutical and papers industries, along with enzyme electrodes for the detection of sucrose.
External RessourcesExternal Ressources
PDB Entry : 1UYP [1]
ReferencesReferences
- ↑ 3.2.1.26 : beta-fructofuranosidase http://www.brenda-enzymes.org/index.php4?page=information/all_enzymes.php4?ecno=
- ↑ Glycoside Hydrolase family classification http://www.cazy.org/Glycoside-Hydrolases.html
- ↑ Glycoside Hydrolase family 32, http://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_32
- ↑ 4.0 4.1 4.2 4.3 4.4 Alberto F, Bignon C, Sulzenbacher G, Henrissat B, Czjzek M. The three-dimensional structure of invertase (beta-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases. J Biol Chem. 2004 Apr 30;279(18):18903-10. Epub 2004 Feb 18. PMID:14973124 doi:10.1074/jbc.M313911200 Cite error: Invalid
<ref>tag; name "Molecular" defined multiple times with different content - ↑ S.M. Kotwal, V. Shankar, Biotechnology Advances, Immobilized invertase, 2009
ContributorsContributors
Sarah Bouteben, Justine Braguy