PDBsum entry 1ob0

Hydrolase

PDB id: 1ob0

Contents

Protein chain

481 a.a. *

Metals

_NA

_CA ×3

Waters ×324

* Residue conservation analysis

PDB id:

1ob0

Name:

Hydrolase

Title:

Kinetic stabilization of bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface

Structure:

Alpha-amylase. Chain: a. Synonym: 1,4-alpha-d-glucan-4-glucanohydrolase. Engineered: yes. Mutation: yes

Source:

Bacillus licheniformis. Organism_taxid: 1402. Expressed in: bacillus subtilis. Expression_system_taxid: 1423

Resolution:

1.83Å R-factor: 0.147 R-free: 0.154

Authors:

M.Machius,N.Declerck,R.Huber,G.Wiegand

Key ref:

M.Machius et al. (2003). Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface. J Biol Chem, 278, 11546-11553. PubMed id: 12540849 DOI: 10.1074/jbc.M212618200

Date:

21-Jan-03 Release date: 30-Jan-03

Protein chain

P06278  (AMY_BACLI) -  Alpha-amylase from Bacillus licheniformis

Seq: 512 a.a.

Struc: 481 a.a.*

* PDB and UniProt seqs differ at 8 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.

DOI no: 10.1074/jbc.M212618200

J Biol Chem 278:11546-11553 (2003)

PubMed id: 12540849

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface.

M.Machius, N.Declerck, R.Huber, G.Wiegand.

ABSTRACT

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.

Selected figure(s)

Figure 2.
Fig. 2. Crystal structure of kinetically stabilized BLA. A, representative 2F[o] F[c] simulated annealing omit electron density map for a region in the core of the kinetically stabilized BLA variant. B, stereoview of a schematic representation of the overall structure of BLA with the mutation sites labeled. Calcium ions are shown in cyan, and sodium is in yellow. All figures were created using the programs Bobscript (54) and POVRray (Persistence of Vision, v3.02, POV-Team, www.povray.org) and GL_RENDER (L. Esser, University of Texas Southwestern Medical Center).

Figure 4.
Fig. 4. Stereoview of the region around the Ca-Na-Ca metal triad containing the mutation N190F. Calcium ions are shown in cyan and the sodium ion is in yellow; other atoms are in standard colors.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 11546-11553) copyright 2003.

Figures were selected by the author.