PDBsum entry: 1jd7

Hydrolase

PDB id: 1jd7

Contents

Protein chain

448 a.a. *

Metals

_CL ×2

_CA

Waters ×385

* Residue conservation analysis

PDB id:

1jd7

Name:

Hydrolase

Title:

Crystal structure analysis of the mutant k300r of pseudoalteromonas haloplanctis alpha-amylase

Structure:

Alpha-amylase. Chain: a. Engineered: yes. Mutation: yes

Source:

Pseudoalteromonas haloplanktis. Organism_taxid: 228. Strain: a23. Gene: amy. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.25Å R-factor: 0.179 R-free: 0.215

Authors:

N.Aghajari,R.Haser

Key ref:

N.Aghajari et al. (2002). Structural basis of alpha-amylase activation by chloride. Protein Sci, 11, 1435-1441. PubMed id: 12021442 DOI: 10.1110/ps.0202602

Date:

13-Jun-01 Release date: 18-Sep-02

Protein chain

P29957  (AMY_PSEHA) -  Alpha-amylase

Seq: 669 a.a.

Struc: 448 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

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 

• Biological process: carbohydrate metabolic process (1 term)
• Biochemical function: catalytic activity (2 terms)

DOI no: 10.1110/ps.0202602

Protein Sci 11:1435-1441 (2002)

PubMed id: 12021442

Structural basis of alpha-amylase activation by chloride.

N.Aghajari, G.Feller, C.Gerday, R.Haser.

ABSTRACT

To further investigate the mechanism and function of allosteric activation by chloride in some alpha-amylases, the structure of the bacterial alpha-amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to alpha-amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal alpha-amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.

Selected figure(s)

Figure 1.
Fig. 1. Schematic representation of the chloride binding site and of the interaction network with active site residues (adapted from Qian et al. 1994). The essential catalytic residues are in italics. Chloride ligands are Lys300(337), Arg172(195), Asn262(298), and H[2]O1003(525); active site residues are Glu200(233), Asp174(197), and Asp264(300) the putative catalytic water molecule H[2]O1004(cat) and His263(299). Numbers in parentheses refer to pig pancreas -amylase.

Figure 2.
Fig. 2. Superposition of the chloride binding site in P. haloplanktis -amylase (AHA), in green, complex AHA/nitrate in blue, mutant K300Q in yellow, and mutant K300R in pink.

The above figures are reprinted by permission from the Protein Society: Protein Sci (2002, 11, 1435-1441) copyright 2002.

Figures were selected by an automated process.