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
(2.4.1.10)
(Sequence Homologues) (PDB Homologues)
- Biological species
-
Bacillus subtilis subsp. subtilis str. 168 (Bacteria)
- PDB
-
1oyg
- Crystal structure of Bacillus subtilis levansucrase
(1.5 Å)
- Catalytic CATH Domains
-
2.115.10.20
(see all for 1oyg)
Enzyme Reaction (EC:2.4.1.10)
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) |
Chemical Components
References
- 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.
- 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.
- 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.
- 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.
- 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.
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 |