title: Site-directed mutations in Alanine 223 and Glycine 255 in the acceptor site of gamma-Cyclodextrin glucanotransferase from Alkalophilic Bacillus clarkii 7364 affect cyclodextrin production.
abstract: A cyclodextrin glucanotransferase (CGTase) from Bacillus clarkii 7364 converts starch into gamma-cyclodextrin (gamma-CD) with high specificity. Comparison of the deduced amino acid sequence of this CGTase with those of other typical CGTases revealed that several amino acids are deleted or substituted with others at several subsites. Of these amino acids, Ala223 at subsite +2 and Gly255 at subsite +3 in the acceptor site of the enzyme were replaced by several amino acids through site-directed mutagenesis. The replacement of Ala223 by lysine, arginine and histidine strongly enhanced the gamma-CD-forming activity in the neutral pH range. On the other hand, all mutants obtained on replacing Gly255 with the above amino acids showed significant decreases in the gamma-CD-forming activity. Taking into account both the kinetic parameters and pKa values of the side chains of the three basic amino acids, the protonation state of the amino groups in their side chains at subsite +2 seems to enhance the hydrogen bonding interaction between these basic amino acids and the glucose residues of linear oligosaccharides. The enhancement of the interaction may play an important role by helping the substrate reach subsite +1, hence increasing the gamma-CD-forming activity and kcat value.
authors.affiliation: Nihon Shokuhin Kako Co., Ltd., 30 Tajima, Fuji 417-8530. yoshinori.nakagawa@jamstec.go.jp
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Nakagawa
authors.forename: Masayasu
authors.initials: M
authors.lastname: Takada
authors.forename: Koichi
authors.initials: K
authors.lastname: Ogawa
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hatada
authors.forename: Koki
authors.initials: K
authors.lastname: Horikoshi
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D047408:gamma-Cyclodextrins / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D047408:gamma-Cyclodextrins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C023792:gamma-cyclodextrin