THE RAW STARCH BINDING DOMAIN OF CYCLODEXTRIN GLYCOSYLTRANSFERASE FROM BACILLUS-CIRCULANS STRAIN-251

The E-domain of cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19 ) from Bacillus circulans strain 251 is a putative raw starch binding domain. Analysis of the maltose-dependent CGTase crystal structure rev ealed that each enzyme molecule contained three maltose molecules, sit uated at contact points between protein molecules. Two of these maltos es were bound to specific sites in the E-domain, the third maltose was bound at the C-domain. To delineate the roles in raw starch binding a nd cyclization reaction kinetics of the two maltose binding sites in t he E-domain, we replaced Trp-616 and Trp-662 of maltose binding site 1 and Tyr-633 of maltose binding site 2 by alanines using site-directed mutagenesis. Purified mutant CGTases were characterized with respect to raw starch binding and cyclization reaction kinetics on both solubl e and raw starch. The results show that maltose binding site 1 is most important for raw starch binding, whereas maltose binding site 2 is i nvolved in guiding linear starch chains into the active site. beta-Cyc lodextrin causes product inhibition by interfering with catalysis in t he active site and the function of maltose binding site 2 in the E-dom ain. CGTase mutants in the E-domain maltose binding site 1 could no lo nger be crystallized as maltose-dependent monomers. Instead, the W616A mutant CGTase protein was successfully crystallized as a carbohydrate -independent dimer; its structure has been refined to 2.2 Angstrom res olution. The three-dimensional structure shows that, within the error limits, neither the absence of carbohydrates nor the W616A mutation ca used significant further conformational changes. The modified starch b inding and cyclization kinetic properties observed with the mutant CGT ase proteins thus can be directly related to the amino acid replacemen ts.