title: Val326 of Thermoactinomyces vulgaris R-47 amylase II modulates the preference for alpha-(1,4)- and alpha-(1,6)-glycosidic linkages.
abstract: Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) catalyzes not only the hydrolysis of alpha-(1,4)- and alpha-(1,6)-glycosidic linkages but also transglycosylation. The subsite +1 structure of alpha-amylase family enzymes plays important roles in substrate specificity and transglycosylation activity. We focused on the amino acid residue at the 326th position based on information on the primary structure and crystal structure, and replaced Val with Ala, Ile, or Thr. The V326A mutant favored hydrolysis of the alpha-(1,4)-glycosidic linkage compared to the wild-type enzyme. In contrast, the V326I mutant favored hydrolysis of the alpha-(1,6)-glycosidic linkage and exhibited low transglycosylation activity. In the case of the V326T mutant, the hydrolytic activity was almost identical to that of the wild-type TVA II, and the transglycosylation activity was poor. These results suggest that the volume and the hydrophobicity of the amino acid residue at the 326th position modulate both the preference for glycosidic linkages and the transglycosylation activity.
authors.affiliation: Department of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ito
authors.forename: Sohei
authors.initials: S
authors.lastname: Ito
authors.forename: Kousuke
authors.initials: K
authors.lastname: Ishino
authors.forename: Akiko
authors.initials: A
authors.lastname: Shimizu-Ibuka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakai
mesh_terms: D000595:Amino Acid Sequence; D006031:Glycosylation; D006868:Hydrolysis; D008848:Micromonosporaceae / Q000201:enzymology; D013379:Substrate Specificity; D014633:Valine / Q000502:physiology*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000516:alpha-Amylases; D014633:Valine