Reference "Crystal and molecular structure of barley alpha-amylase"

Reference ID

8588

Title

Crystal and molecular structure of barley alpha-amylase

Source

Journal of molecular biology, 1994, vol. 239, pp. 104-121

Authors (4)

Kadziola-A	Abe-J
Svensson-B	Haser-R

Abstract

The three-dimensional structure of barley malt alpha-amylase (isoform AMY2-2)
was determined by multiple isomorphous replacement using three heavy-atom
derivatives and solvent flattening. The model was refined using a combination of
simulated annealing and conventional restrained least-squares crystallographic
refinement to an R-factor of 0.153 based on 18,303 independent reflections with
F(o) > sigma(F(o)) between 10 and 2.8 A resolution, with root-mean-square
deviations of 0.016 A and 3.3 degrees from ideal bond lengths and bond angles,
respectively. The final model consists of 403 amino acid residues, three calcium
ions and 153 water molecules. The polypeptide chain folds into three domains: a
central domain forming a (beta alpha)8-barrel of 286 residues, with a protruding
irregular structured loop domain of 64 residues (domain B) connecting strand
beta 3 and helix alpha 3 of the barrel, and a C-terminal domain of 53 residues
forming a five stranded anti-parallel beta-sheet. Unlike the previously known
alpha-amylase structures, AMY2-2 contains three Ca2+ binding sites co-ordinated
by seven or eight oxygen atoms from carboxylate groups, main-chain carbonyl
atoms and water molecules, all calcium ions being bound to domain B and
therefore essential for the structural integrity of that domain. Two of the Ca2+
sites are located only 7.0 A apart with one Asp residue serving as ligand for
both. One Ca2+ site located at about 20 A from the other two was found to be
exchangeable with Eu3+. By homology with other alpha-amylases, some important
active site residues are identified as Asp179, Glu204 and Asp289, and are
situated at the C-terminal end of the central beta-barrel. A starch granule
binding site, previously identified as Trp276 and Trp277, is situated on alpha-
helix 6 in the central (beta alpha)8-barrel, at the surface of the enzyme. This
binding site region is associated with a considerable disruption of the (beta
alpha)8-barrel 8-fold symmetry.