4-alpha-glucanotransferase
Amylomaltase enzymes are structurally and mechanistically related to alpha amylases, although they almost exclusively catalyse trans-glycosylation reactions whereas alpha amylase enzymes catalyse hydrolysis reactions. The amylomaltase enzyme catalyses amylose disproportionation and the synthesis of large cyclic glucans, making these enzymes of interest to chemical and pharmaceutical industries.
Reference Protein and Structure
- Sequence
-
O87172
(2.4.1.25)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Thermus thermophilus (Bacteria)
- PDB
-
1cwy
- CRYSTAL STRUCTURE OF AMYLOMALTASE FROM THERMUS AQUATICUS, A GLYCOSYLTRANSFERASE CATALYSING THE PRODUCTION OF LARGE CYCLIC GLUCANS
(2.0 Å)
- Catalytic CATH Domains
-
3.20.20.80
(see all for 1cwy)
Enzyme Reaction (EC:2.4.1.25)
Enzyme Mechanism
Introduction
Three conserved carboxylic acid residues are central to the catalytic mechanism. The first acid residue acts as a general acid towards the oxygen of the scissile glycosidic bond. Simultaneously, the oxygen of a second carboxylic residue acts as a nucleophile towards the C1 of the substrate. This forms a covalent glycosyl-enzyme intermediate though a planar oxocarbenium intermediate. This covalent intermediate can be broken down by either a water molecule, resulting in hydrolysis or by a hydroxyl group of another sugar molecule. The third catalytic carboxylic residue binds the sugar in the -1 subsite, distorting it towards a partially planar conformation and contributing to the transition state stabilisation through hydrogen bonding.
Catalytic Residues Roles
| UniProt | PDB* (1cwy) | ||
| Asp293 | Asp293A | The residue acts as a nucleophile to the C1 of the scissile glycosidic bond in a SN2 displacement mechanism, resulting in the formation of covalent glycosyl-enzyme intermediate. The residue is released from the intermediate by SN2 displacement at the C1 from an activated second sugar moiety. | covalent catalysis |
| Glu340 | Glu340A | The residue acts as a general acid to the oxygen of the glycosidic bond, enhancing nucleophilic attack at the substrate's C1 position. It then acts as a general base towards the attacking sugar molecule in the collapse of the glycosyl-enzyme intermediate and formation of a new glycosidic link. In hydrolysis, this residue activates a water molecule towards hydrolysis at the glycosidic bond. It is interesting to note that the orientation of Glu340 is optimised as to reduce water activation, protecting the intermediate from hydrolysis. This fits with the experimental observation that the Thermus enzyme is 5000 times less likely to utilise water in hydrolysis than maltotriose in glucan formation. |
proton shuttle (general acid/base) |
| Asp395 | Asp395A | The residue binds to the sugar substrate in the -1 subsite by hydrogen boding to the 2- and 3-OH groups, distorting its structure towards a planar conformation and stabilising the reaction transition state. | electrostatic stabiliser |
Chemical Components
References
- Barends TR et al. (2007), J Biol Chem, 282, 17242-17249. Three-way Stabilization of the Covalent Intermediate in Amylomaltase, an -Amylase-like Transglycosylase. DOI:10.1074/jbc.m701444200. PMID:17420245.
- Roth C et al. (2017), Sci Adv, 3, e1601386-. Amylose recognition and ring-size determination of amylomaltase. DOI:10.1126/sciadv.1601386. PMID:28097217.
- Fujii K et al. (2007), J Biosci Bioeng, 103, 167-173. Function of second glucan binding site including tyrosines 54 and 101 in Thermus aquaticus amylomaltase. DOI:10.1263/jbb.103.167. PMID:17368400.
- Przylas I et al. (2000), J Mol Biol, 296, 873-886. Crystal structure of amylomaltase from Thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans. DOI:10.1006/jmbi.1999.3503. PMID:10677288.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Glu340A | proton shuttle (general acid/base) |
| Asp395A | electrostatic stabiliser |
| Asp293A | covalent catalysis |