Mutagenesis and structural analysis of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II)

An alpha-amylase II (TVA II) produced by Thermoactinomyces vulgaris R-47 exhibits wide substrate specificity for starch, pullulan, cyclodextrins and isopanose. Asp465 and Arg469 are located at subsite (- 2) and are observed among many alpha-amylase family enzymes. Here, we have investigated the rela...

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Bibliographic Details
Published in:Journal of Applied Glycoscience 2005, Vol.52(3), pp.225-231
Main Authors: Mizuno, M.(Tokyo Univ. of Agriculture and Technology, Fuchu (Japan)), Ichikawa, K, Tonozuka, T, Ohtaki, A, Shimura, Y, Kamitori, S, Nishikawa, A, Sakano, Y
Format: Article
Language:English
Subjects:
ACIDE ASPARTIQUE
ACIDO ASPARTICO
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
ALFA AMILASA
ALMIDON
ALPHA AMYLASE
AMIDON
ARGININA
ARGININE
ASPARTIC ACID
ENZYME ACTIVITY
ESCHERICHIA COLI
MUTACION
MUTATION
OLIGOSACARIDOS
OLIGOSACCHARIDE
OLIGOSACCHARIDES
RECOMBINACION
RECOMBINAISON
RECOMBINATION
STARCH
subsite
substrate recognition
THERMOACTINOMYCES
Thermoactinomyces vulgaris
TVA II
α-amylase family
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Summary:An alpha-amylase II (TVA II) produced by Thermoactinomyces vulgaris R-47 exhibits wide substrate specificity for starch, pullulan, cyclodextrins and isopanose. Asp465 and Arg469 are located at subsite (- 2) and are observed among many alpha-amylase family enzymes. Here, we have investigated the relationship between the substrate specificity of TVA II and the substrate binding by Asp465 and Arg469 using site-directed mutagenesis and X-ray crystallographic analyses. The five mutated TVA IIs (D465N, D465E, D465Q, R469L and R469K) showed lower specific activities for all substrates tested here than the wild-type TVA II. In particular, the mutation of Arg469 significantly decreased the hydrolyzing activities compared to the mutation of Asp465. While the Km values for alpha-cyclodextrin were ipcreased for all the mutants, large decreases of the k(cat) values were observed only for R469L and R469K. The side chain of Arg469 is close to that of Asp421, which is one of three catalytic residues, and the positive charge of Arg469 seems to affect the catalytic mechanism via Asp421. These findings suggest that Asp465 and Arg469 are important residues for the substrate binding and the catalytic reaction. Furthermore, the binding of the alpha-maltosyl unit by Asp465 and Arg469 is commonly observed among TVA II and other alpha-amylase family enzymes, which is thought to be the basic mechanism of substrate binding.
ISSN:1344-7882
1880-7291
DOI:10.5458/jag.52.225