Neopullulanase

 

Neopullulanase is a member of the glycoside hydrolase family 13 (GH13) and is one of the family enzymes with a unique substrate specificity that hydrolyses pullulan to produce panose, maltose and glucose in a final molar ratio of 3:1:1. This enzyme hydrolyses alpha-1,4 and alpha-1,6-glucosidic linkages, and catalyses transglycosylation reactions to form both alpha-1,4 and alpha-1,6-glucosidic bonds.

Neopullulanase (EC 3.2.1.135) is a special glycoside hydrolase that is able to catalyse several biochemical reactions, such as the hydrolysis of both alpha-1,4- and alpha-1,6-glucosidic linkages and their formation by transglycosylation. Neopullulanase is, in fact, nearly indistinguishable from the two closely related enzyme specificities, maltogenic amylase (EC 3.2.1.133) and cyclomaltodextrinase (EC 3.2.1.54). The three groups of enzymes contain ∼130 residues at the N terminus that are absent in the smaller alpha-amylases.

They are known to bind Ca, but it is remote from the active site and not involved in the mechanism.

 

Reference Protein and Structure

Sequence
Q08751 UniProt (3.2.1.135) IPR006047 (Sequence Homologues) (PDB Homologues)
Biological species
Thermoactinomyces vulgaris (Bacteria) Uniprot
PDB
1bvz - ALPHA-AMYLASE II (TVAII) FROM THERMOACTINOMYCES VULGARIS R-47 (2.6 Å) PDBe PDBsum 1bvz
Catalytic CATH Domains
3.20.20.80 CATHdb (see all for 1bvz)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.135)

pullulan
CHEBI:27941ChEBI
+
water
CHEBI:15377ChEBI
→
panose
CHEBI:7912ChEBI
+
pullulan
CHEBI:27941ChEBI
Alternative enzyme names: Pullulanase II,

Enzyme Mechanism

Introduction

Has a very similar mechanism to other alpha-amylases: Asp325 acts as a nucleophile, whereas Glu354 acts as an acid-base. Asp421 stabilises the transition state. Glu354 initiates the reaction by protonating O4 atom, giving an oxocarbonium ion-like transition state. Asp325 makes a nucleophilic attack on the C1 atom to form glycosyl-enzyme intermediate. Glu354 acts as a general base to activate a water. The activated water attacks on the intermediate to complete the hydrolysis.

Catalytic Residues Roles

UniProt PDB* (1bvz)
Asp325 Asp325A Acts as a nucleophile. covalent catalysis
Glu354 Glu354A Acts as a general acid/base. proton shuttle (general acid/base)
Asp421 Asp421A Stabilises the transition states. transition state stabiliser
*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. MacGregor E et al. (2001), Biochim Biophys Acta Protein Struct Mol Enzymol, 1546, 1-20. Relationship of sequence and structure to specificity in the α-amylase family of enzymes. DOI:10.1016/s0167-4838(00)00302-2.
  2. Kumar V (2011), Bioinformation, 6, 61-63. Identification of the sequence motif of glycoside hydrolase 13 family members. PMID:21544166.
  3. Ohtaki A et al. (2004), J Biol Chem, 279, 31033-31040. Complex Structures of Thermoactinomyces vulgaris R-47  -Amylase 2 with Acarbose and Cyclodextrins Demonstrate the Multiple Substrate Recognition Mechanism. DOI:10.1074/jbc.m404311200. PMID:15138257.
  4. Hondoh H et al. (2003), J Mol Biol, 326, 177-188. Three-dimensional Structure and Substrate Binding of Bacillus stearothermophilus Neopullulanase. DOI:10.1016/s0022-2836(02)01402-x.
  5. Kamitori S et al. (2002), J Mol Biol, 318, 443-453. Crystal Structures and Structural Comparison of Thermoactinomyces vulgaris R-47 α-Amylase 1 (TVAI) at 1.6Å Resolution and α-Amylase 2 (TVAII) at 2.3Å Resolution. DOI:10.1016/s0022-2836(02)00111-0. PMID:12051850.
  6. Lee HS et al. (2002), J Biol Chem, 277, 21891-21897. Cyclomaltodextrinase, Neopullulanase, and Maltogenic Amylase Are Nearly Indistinguishable from Each Other. DOI:10.1074/jbc.m201623200. PMID:11923309.
  7. Park K et al. (2000), Biochim Biophys Acta Protein Struct Mol Enzymol, 1478, 165-185. Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the α-amylase family. DOI:10.1016/s0167-4838(00)00041-8.
  8. Svensson B (1994), Plant Mol Biol, 25, 141-157. Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability. PMID:8018865.

Step 1.

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Catalytic Residues Roles

Residue Roles
Asp325A covalent catalysis
Glu354A proton shuttle (general acid/base)
Asp421A transition state stabiliser

Chemical Components

Step 1.

Contributors

Gemma L. Holliday, Nozomi Nagano, Craig Porter