PDBsum entry: 2qpu

Hydrolase

PDB id: 2qpu

Contents

Protein chains

404 a.a. *

Ligands

QPU ×4

EDO ×2

QPS

BGC

Metals

_CA ×12

Waters ×1726

* Residue conservation analysis

PDB id:

2qpu

Name:

Hydrolase

Title:

Sugar tongs mutant s378p in complex with acarbose

Structure:

Alpha-amylase type a isozyme. Chain: a, b, c. Synonym: 1,4-alpha-d- glucan glucanohydrolase, amy1, low pi amylase. Engineered: yes. Mutation: yes

Source:

Hordeum vulgare. Barley. Organism_taxid: 4513. Gene: amy1.1. Expressed in: pichia pastoris. Expression_system_taxid: 4922

Resolution:

1.70Å R-factor: 0.178 R-free: 0.209

Authors:

N.Aghajari,M.H.Jensen,S.Tranier,R.Haser

Key ref:

S.Bozonnet et al. (2007). The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity. Febs J, 274, 5055-5067. PubMed id: 17803687 DOI: 10.1111/j.1742-4658.2007.06024.x

Date:

25-Jul-07 Release date: 01-Jul-08

Protein chains

P00693  (AMY1_HORVU) -  Alpha-amylase type A isozyme

Seq: 438 a.a.

Struc: 404 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.1 - Alpha-amylase.
• Reaction: Endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides.

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (2 terms)
• Biological process: metabolic process (2 terms)
• Biochemical function: catalytic activity (7 terms)

DOI no: 10.1111/j.1742-4658.2007.06024.x

Febs J 274:5055-5067 (2007)

PubMed id: 17803687

The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity.

S.Bozonnet, M.T.Jensen, M.M.Nielsen, N.Aghajari, M.H.Jensen, B.Kramhøft, M.Willemoës, S.Tranier, R.Haser, B.Svensson.

ABSTRACT

Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as K(d) = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates.