PDBsum entry: 1rp9

Hydrolase

PDB id: 1rp9

Contents

Protein chain

404 a.a. *

Ligands

DAF-BGC-GLC

DAF-GLC-GLC ×2

Metals

_CA ×3

Waters ×396

* Residue conservation analysis

PDB id:

1rp9

Name:

Hydrolase

Title:

Crystal structure of barley alpha-amylase isozyme 1 (amy1) inactive mutant d180a in complex with acarbose

Structure:

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

Source:

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

Resolution:

2.00Å R-factor: 0.166 R-free: 0.226

Authors:

X.Robert,R.Haser,N.Aghajari

Key ref:

X.Robert et al. (2005). Oligosaccharide binding to barley alpha-amylase 1. J Biol Chem, 280, 32968-32978. PubMed id: 16030022 DOI: 10.1074/jbc.M505515200

Date:

03-Dec-03 Release date: 07-Jun-05

Protein chain

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: 10.1074/jbc.M505515200

J Biol Chem 280:32968-32978 (2005)

PubMed id: 16030022

Oligosaccharide binding to barley alpha-amylase 1.

X.Robert, R.Haser, H.Mori, B.Svensson, N.Aghajari.

ABSTRACT

Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."

Selected figure(s)

Figure 2.
FIGURE 2. A, atoms labeling convention for glucosyl residues and acarbose. The acarviosine unit is constituted by rings A and B, including the amino group valienamine, and are -1,4-linked to rings C and D representing a maltose unit. B, superimposition of residues implicated in the substrate binding in active sites of AMY1 and AMY2 (stereo view). Only residues interacting directly by hydrogen bonds are shown. The complex AMY2-acarbose (4) (Protein Data Bank entry 1BG9 [PDB] ) is presented in blue, AMY1-acarbose (this work, Protein Data Bank entry 1RPK [PDB] ) in red, AMY1-thio-DP4 (28) (Protein Data Bank entry 1P6W [PDB] ) in green, and AMY1[D180A]-acarbose (this work, Protein Data Bank entry 1RP9 [PDB] ) in yellow.

Figure 4.
FIGURE 4. Schematic representation of hydrogen bonding network in the catalytic cleft of the AMY1[D180A]-maltoheptaose complex. Amino acid residues are in rectangles and water molecules in ellipsoids. Catalytic residues are highlighted. Figure was rendered using the ISIS-Draw software.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 32968-32978) copyright 2005.

Figures were selected by an automated process.