title: Complexes of Thermoactinomyces vulgaris R-47 alpha-amylase 1 and pullulan model oligossacharides provide new insight into the mechanism for recognizing substrates with alpha-(1,6) glycosidic linkages.
abstract: Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of alpha-(1,4), alpha-(1,4), and alpha-(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes alpha-(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes alpha-(1,6) glycosidic linkages with a lesser efficiency. X-ray structures of three complexes comprising an inactive mutant TVAI (D356N or D356N/E396Q) and a pullulan model oligosaccharide (P2; [Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]2 or P5; [Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]5) were determined. The complex D356N/P2 is a mimic of the enzyme/product complex in the main catalytic reaction of TVAI, and a structural comparison with Aspergillus oryzaealpha-amylase showed that the (-) subsites of TVAI are responsible for recognizing both starch and pullulan. D356N/E396Q/P2 and D356N/E396Q/P5 provided models of the enzyme/substrate complex recognizing the alpha-(1,6) glycosidic linkage at the hydrolyzing site. They showed that only subsites -1 and -2 at the nonreducing end of TVAI are effective in the hydrolysis of alpha-(1,6) glycosidic linkages, leading to weak interactions between substrates and the enzyme. Domain N of TVAI is a starch-binding domain acting as an anchor in the catalytic reaction of the enzyme. In this study, additional substrates were also found to bind to domain N, suggesting that domain N also functions as a pullulan-binding domain.
authors.affiliation: Graduate School of Medicine, Kagawa University, Japan.
authors.forename: Akemi
authors.initials: A
authors.lastname: Abe
authors.forename: Hiromi
authors.initials: H
authors.lastname: Yoshida
authors.forename: Takashi
authors.initials: T
authors.lastname: Tonozuka
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Sakano
authors.forename: Shigehiro
authors.initials: S
authors.lastname: Kamitori
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D008848:Micromonosporaceae / Q000737:chemistry / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D046912:Multiprotein Complexes; D009844:Oligosaccharides; C009109:pullulan; D000516:alpha-Amylases