Mutual conversion of substrate specificities of Thermoactinomyces vulgaris R-47 alpha-amylases TVAI and TVAII by site-directed mutagenesis.
Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVAI and TVAII, differing in substrate specificity from each other. TVAI favors high-molecular-weight substrates like starch, and scarcely hydrolyzes cyclomaltooligosaccharides (cyclodextrins) with a small cavity. TVAII favors low-molecular-weight substrates like oligosaccharides, and can efficiently hydrolyze cyclodextrins with various sized cavities. To understand the relationship between the structure and substrate specificity of these enzymes, we precisely examined the roles of key residues for substrate recognition by X-ray structural and kinetic parameter analyses of mutant enzymes and successfully obtained mutants in which the substrate specificity of each enzyme is partially converted into that of another.
Ohtaki
Akashi
A
Department of Biotechnology and Life Science, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Iguchi
Akihiro
A
Mizuno
Masahiro
M
Tonozuka
Takashi
T
Sakano
Yoshiyuki
Y
Kamitori
Shigehiro
S
D001665:Binding Sites; D018360:Crystallography, X-Ray; D007700:Kinetics; D008848:Micromonosporaceae / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000516:alpha-Amylases