1OB0

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

Summary for 1OB0

• Entry DOI: 10.2210/pdb1ob0/pdb
• Related: 1BLI 1BPL 1E3X 1E3Z 1E40 1E43 1VJS
• Descriptor: ALPHA-AMYLASE, CALCIUM ION, SODIUM ION, ... (4 entities in total)
• Functional Keywords: hydrolase, glycosyltransferase, starch degradation, thermostability, calcium, sodium
• Biological source: BACILLUS LICHENIFORMIS
• Total number of polymer chains: 1
• Total formula weight: 55457.25

Authors

Machius, M.,Declerck, N.,Huber, R.,Wiegand, G. (deposition date: 2003-01-21, release date: 2003-01-30, Last modification date: 2023-12-13)

Primary citation

Machius, M.,Declerck, N.,Huber, R.,Wiegand, G.
Kinetic Stabilization of Bacillus Licheniformis Alpha-Amylase Through Introduction of Hydrophobic Residues at the Surface
J.Biol.Chem., 278:11546-, 2003

PubMed 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.

PubMed: 12540849
DOI: 10.1074/JBC.M212618200

Experimental method

X-RAY DIFFRACTION (1.83 Å)