title: Role of Phe286 in the recognition mechanism of cyclomaltooligosaccharides (cyclodextrins) by Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII). X-ray structures of the mutant TVAIIs, F286A and F286Y, and kinetic analyses of the Phe286-replaced mutant TVAIIs.
abstract: Phe286 located in the center of the active site of alpha-amylase 2 from Thermoactinomyces vulgaris R-47 (TVAII) plays an important role in the substrate recognition for cyclomaltooligosaccharides (cyclodextrins). The X-ray structures of mutant TVAIIs with the replacement of Phe286 by Ala (F286A) and Tyr (F286Y) were determined at 3.2 A resolution. Their structures have no significant differences from that of the wild-type enzyme. The kinetic analyses of Phe286-replaced variants showed that the variants with non-aromatic residues, Ala (F286A) and Leu (F286L), have lower enzymatic activities than those with aromatic residues, Tyr (F286Y) and Trp (F286W), and the replacement of Phe286 affects enzymatic activities for CDs more than those for starch.
authors.affiliation: Department of Biotechnology and Life Science, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, 184-8588, Tokyo, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Ohtaki
authors.forename: S
authors.initials: S
authors.lastname: Kondo
authors.forename: Y
authors.initials: Y
authors.lastname: Shimura
authors.forename: T
authors.initials: T
authors.lastname: Tonozuka
authors.forename: Y
authors.initials: Y
authors.lastname: Sakano
authors.forename: S
authors.initials: S
authors.lastname: Kamitori
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D003505:Cyclodextrins / Q000737:chemistry*; D007700:Kinetics; D008848:Micromonosporaceae / Q000201:enzymology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D003505:Cyclodextrins; D014443:Tyrosine; D010649:Phenylalanine; D000516:alpha-Amylases; D000409:Alanine