Three-dimensional structure of Pseudomonas isoamylase at 2.2 Å resolution

The three-dimensional structure of isoamylase from Pseudomonas amyloderamosa, which hydrolyzes alpha-1,6-glucosidic linkages of amylopectin and glycogen, has been determined by X-ray structure analysis. The enzyme has 750 amino acid residues and a molecular mass of 80 kDa, and it can be crystallized from ammonium sulfate solution. The structure was elucidated by the multiple isomorphous replacement method and refined at 2.2 Angstrom resolution, resulting in a final R-factor of 0.161 for significant reflections with a root-mean-square deviation from ideality in bond lengths of 0.009 Angstrom. The analysis revealed that in the N-terminal region, isoamylase has a novel extra domain that we call domain N, whose three-dimensional structure has not so far been reported. It has a (beta/alpha)(8)-barrel-type supersecondary structure in the catalytic domain common to the alpha-amylase family enzymes, though the barrel is incomplete, with a deletion of an alpha-helix between the fifth and sixth beta-strands. A long excursed region is present between the third beta-strand and the third alpha-helix of the barrel but, in contrast to the so-called domain B that has been identified in the other enzymes of alpha-amylase family, it cannot be considered to be an independent domain, because this loop forms a globular cluster together with the loop between the fourth beta-strand and the fourth alpha-helix. Isoamylase contains a bound calcium ion, but this is not in the same position as the conserved calcium ion that has been reported in other alpha-amylase family enzymes. (C) 1998 Academic Press.