Analysis of catalytic residues of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) by site-directed mutagenesis.
To confirm that the catalytic residues (Asp325, Glu354, and Asp421) are necessary for the hydrolysis of starch, pullulan, and cyclodextrins, we constructed TVA II mutated by site-directed mutagenesis. The mutated enzymes (D325N, E354Q, and D421N) had markedly reduced levels of activity, less than 0.006% of the wild type, indicating that these three residues are the catalytic sites for these substrates. Even E354D had reduced levels of activity, less than 0.05% of wild type. These four mutated enzymes retained a trace of activity. From the result of hydrolysis patterns for maltohexaose, in particular, D421N, unlike D325N and E354Q, catalyzed transglycosylation rather than hydrolysis. The results suggest that Asp421 could function to capture water molecules.
Ichikawa
K
K
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Japan.
Tonozuka
T
T
Yokota
T
T
Shimura
Y
Y
Sakano
Y
Y
D020134:Catalytic Domain; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D008320:Maltose / Q000378:metabolism; D008848:Micromonosporaceae / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
D009844:Oligosaccharides; C016549:maltohexaose; D008320:Maltose; D000516:alpha-Amylases; D005947:Glucose