title: The residue 179 is involved in product specificity of the Bacillus circulans DF 9R cyclodextrin glycosyltransferase.
abstract: Cyclodextrin glycosyltransferases (CGTases) are important enzymes in biotechnology because of their ability to produce cyclodextrin (CD) mixtures from starch whose relative composition depends on enzyme source. A multiple alignment of 46 CGTases and Shannon entropy analysis allowed us to find differences and similarities that could be related to product specificity. Interestingly, position 179 has Gly in all the CGTases except in that from Bacillus circulans DF 9R which possesses Gln. The absence of a side chain at that position has been considered as a strong requirement for substrate binding and cyclization process. Therefore, we constructed two mutants of this enzyme, Q179L and Q179G. The activity and kinetic parameters of Q179G remained unchanged while the Q179L mutant showed a different CDs ratio, a lower catalytic efficiency, and a decreased ability to convert starch into CDs. We show that position 179 is involved in CGTase product specificity and must be occupied by Gly--without a side chain--or by amino acid residues able to interact with the substrate through hydrogen bonds in a way that the cyclization process occurs efficiently. These findings are also explained on the basis of a structural model.
authors: Departamento de Ciencias Básicas, Universidad Nacional de Luján, Rutas 5 y 7 (6700), Luján, Buenos Aires, Argentina.
authors: Hernán
authors: H
authors: Costa
authors: Ana Julia
authors: AJ
authors: Distéfano
authors: Cristina
authors: C
authors: Marino-Buslje
authors: Aurelio
authors: A
authors: Hidalgo
authors: José
authors: J
authors: Berenguer
authors: Mirtha
authors: M
authors: Biscoglio de Jiménez Bonino
authors: Susana Alicia
authors: SA
authors: Ferrarotti
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase