Three-dimensional structure of cyclodextrin glycosyltransferase from Bacillus circulans at 3.4 A resolution.

Cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans has been purified, crystallized and analyzed by X-ray diffraction. The enzyme is monomeric. SDS/polyacrylamide gel electrophoresis gave an Mr of 73,600(+/- 1000), corresponding to 670(+/- 10) amino acid residues. The structure of the crystalline enzyme has been elucidated at a resolution of 3.4 A, using multiple isomorphous replacement and solvent flattening for phase determination. The resulting electron density map allowed tracing of the polypeptide chain; 664 residue positions have been assigned. The chain fold has been subdivided into five domains. The N-terminal domain forms a (beta alpha)8-barrel, which contains the second domain of about 55 residues as an insert after the third beta-strand. The three remaining domains form almost exclusively beta-pleated sheet structures and consist of about 90, 80 and 95 residues. The chain fold of the three N-terminal domains of 492 residues resembles closely the two known structures of alpha-amylases. This geometric similarity corresponds to the observed amino acid sequence homology. On the basis of the sequence homology with alpha-amylases, the active center can be located. The fourth domain has an immunoglobulin fold and is far away from the active center, while the fifth domain participates in the formation of the broad depression at the active center. Accordingly, the cyclodextrin glycosyltransferase chain fold can be considered as an alpha-amylase chain fold with two additional domains.