Levansucrase

 

Member of the glycosyl hydrolase 68 family. Catalyses the polymerisation of fructan via the direct incorporation of a (2,6)-linked furan from sucrose.

 

Reference Protein and Structure

Sequence
P05655 UniProt (2.4.1.10) IPR003469 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus subtilis subsp. subtilis str. 168 (Bacteria) Uniprot
PDB
1oyg - Crystal structure of Bacillus subtilis levansucrase (1.5 Å) PDBe PDBsum 1oyg
Catalytic CATH Domains
2.115.10.20 CATHdb (see all for 1oyg)
Click To Show Structure

Enzyme Reaction (EC:2.4.1.10)

[(2->6)-beta-D-fructofuranosyl-]n alpha-D-glucopyranoside
CHEBI:134464ChEBI
+
sucrose
CHEBI:17992ChEBI
→
D-glucopyranose
CHEBI:4167ChEBI
+
6-kestotriose
CHEBI:64833ChEBI
Alternative enzyme names: Beta-2,6-fructan:D-glucose 1-fructosyltransferase, Beta-2,6-fructosyltransferase, Sucrose 6-fructosyltransferase, Sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, Sucrose:(2->6)-beta-D-fructan 6-beta-D-fructosyltransferase,

Enzyme Mechanism

Introduction

The canonical reaction mechanism of retaining glycosidases implies that Asp86 and Glu342 need to be in different protonation states upon substrate binding in the double displacement reaction mechanism of fructosyl transfer. The optimum of levansucrase enzymatic activity is at pH 6.0. During the first reaction step of the double displacement mechanism, nucleophilic Asp86 forms a covalent intermediate with the fructofuranosyl, while Glu342 protonates the glucosyl leaving group. In the second reaction step of the double displacement mechanism, the acceptor substrate binds and, through nucleophilic attack of the terminal 6'-hydroxyl (assuming a polymer with β (2 → 6) linkage) on the anomeric carbon, the enzyme-bound fructosyl is added to the acceptor.

Catalytic Residues Roles

UniProt PDB* (1oyg)
Ser164 Ser164(138)A Is an important residue to maintain the nucleophile position in the active site. steric locator
Asp86 Asp86(60)A Acts as a nucleophile. covalent catalysis
Asp247 Asp247(221)A Acts to stabilise the reactive intermediates and transition states formed during the course of the reaction. Asp247 forms strong hydrogen bonding interactions with the C3' and C4' hydroxyls of the fructosyl unit. electrostatic stabiliser
Glu342 Glu342(316)A Acts as a general acid/base. proton shuttle (general acid/base)
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Meng G et al. (2008), BMC Struct Biol, 8, 16-. Donor substrate recognition in the raffinose-bound E342A mutant of fructosyltransferase Bacillus subtilis levansucrase. DOI:10.1186/1472-6807-8-16. PMID:18366639.
  2. Raga-Carbajal E et al. (2016), Glycobiology, 26, 377-385. Size product modulation by enzyme concentration reveals two distinct levan elongation mechanisms in Bacillus subtilis levansucrase. DOI:10.1093/glycob/cwv112. PMID:26646447.
  3. Ortiz-Soto ME et al. (2008), Protein Eng Des Sel, 21, 589-595. Selected mutations in Bacillus subtilis levansucrase semi-conserved regions affecting its biochemical properties. DOI:10.1093/protein/gzn036. PMID:18596022.
  4. Seibel J et al. (2006), Carbohydr Res, 341, 2335-2349. Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase). DOI:10.1016/j.carres.2006.07.001. PMID:16870166.
  5. Meng G et al. (2003), Nat Struct Biol, 10, 935-941. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. DOI:10.1038/nsb974. PMID:14517548.

Step 1.

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Catalytic Residues Roles

Residue Roles
Asp86(60)A covalent catalysis
Asp247(221)A electrostatic stabiliser
Glu342(316)A proton shuttle (general acid/base)
Ser164(138)A steric locator

Chemical Components

Step 1.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday