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Literature summary extracted from

  • Mizuno, M.; Tonozuka, T.; Uechi, A.; Ohtaki, A.; Ichikawa, K.; Kamitori, S.; Nishikawa, A.; Sakano, Y.
    The crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) complexed with transglycosylated product (2004), Eur. J. Biochem., 271, 2530-2538.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.1 expresion of mutant enzymes in Escherichai coli strain MV1184 Thermoactinomyces vulgaris
3.2.1.54 expression of mutant enzymes in Escherichai coli strain MV1184 Thermoactinomyces vulgaris

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.2.1.1 purified recombinant TVAII mutant enzyme D325N complexed with alpha-panosylpanose, hanging drop vapour diffusion method, 0.0015 ml of 20 mg/ml protein in 5 m Tris-HCl, pH 7.5, is mixed with 0.0015 ml of reservoir solution containing 1% w/v PEG 6000, 5 mM CaCl2, and 40 mM MES-NaOH, pH 6.1, soaking of crystals in cryoprotection solution containing 20% v/v 2-methyl-2,4-pentanediol, 20% w/v PEG 6000, and 2.5 mM CaCl2, X-ray diffraction structure determination and analysis at 2.2 A resolution, modelling Thermoactinomyces vulgaris
3.2.1.54 purified recombinant TVAII mutant enzyme D325N complexed with alpha-panosylpanose, hanging drop vapour diffusion method, 0.0015 ml of 20 mg/ml protein in 5 m Tris-HCl, pH 7.5, is mixed with 0.0015 ml of reservoir solution containing 1% w/v PEG 6000, 5 mM CaCl2, and 40 mM MES-NaOH, pH 6.1, soaking of crystals in cryoprotection solution containing 20% v/v 2-methyl-2,4-pentanediol, 20% w/v PEG 6000, and 2.5 mM CaCl2, X-ray diffraction structure determination and analysis at 2.2 A resolution, modelling Thermoactinomyces vulgaris

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.1 D325N site-directed mutagenesis, inactive mutant Thermoactinomyces vulgaris
3.2.1.1 Y374A site-directed mutagenesis, the mutant shows reduced activity, and altered substrate specificity and kinetics compared to the wild-type enzyme Thermoactinomyces vulgaris
3.2.1.54 D325N site-directed mutagenesis, inactive mutant Thermoactinomyces vulgaris
3.2.1.54 Y374A site-directed mutagenesis, the mutant shows reduced activity, and altered substrate specificity and kinetics compared to the wild-type enzyme Thermoactinomyces vulgaris

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.1 additional information
-
 additional information kinetic study Thermoactinomyces vulgaris
3.2.1.1 0.19
-
 starch mutant Y374A Thermoactinomyces vulgaris
3.2.1.1 0.23
-
 starch wild-type enzyme Thermoactinomyces vulgaris
3.2.1.1 0.57
-
 pullulan mutant Y374A Thermoactinomyces vulgaris
3.2.1.1 1.4
-
 pullulan wild-type enzyme Thermoactinomyces vulgaris
3.2.1.54 additional information
-
 additional information kinetic study Thermoactinomyces vulgaris
3.2.1.54 0.19
-
 starch mutant Y374A Thermoactinomyces vulgaris
3.2.1.54 0.23
-
 starch wild-type enzyme Thermoactinomyces vulgaris
3.2.1.54 0.57
-
 pullulan mutant Y374A Thermoactinomyces vulgaris
3.2.1.54 1.4
-
 pullulan wild-type enzyme Thermoactinomyces vulgaris

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.1 Thermoactinomyces vulgaris
-
-
-
3.2.1.1 Thermoactinomyces vulgaris R-47
-
-
-
3.2.1.54 Thermoactinomyces vulgaris
-
-
-
3.2.1.54 Thermoactinomyces vulgaris R-47
-
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
3.2.1.1 (alpha-D-glucopyranosyl-(1-4))n-alpha-D-glucopyranose + H2O = (alpha-D-glucopyranosyl-(1-4))n-m-alpha-D-glucopyranose + (alpha-D-glucopyranosyl-(1-4))m-alpha-D-glucopyranose TVAII is a bifunctional enzyme showing alpha-amylase as well as cyclodextrin-hydrolyzing activity, the enzyme hydrolyzes alpha-1,4-glucosidic linkages and alpha-1,6-glucosidic linkages, active site structure and substrate binding structure, Trp356 is involved in substrate binding, and Tyr374 is involved in substrate orientation for catalysis Thermoactinomyces vulgaris
a
3.2.1.54 cyclomaltodextrin + H2O = linear maltodextrin TVA II is a bifunctional enzyme showing alpha-amylase as well as cyclodextrin-hydrolyzing activity, the enzyme hydrolyzes alpha-1,4-glucosidic linkages and alpha-1,6-glucosidic linkages, active site structure and substrate binding structure, Trp356 is involved in substrate binding, and Tyr374 is involved in substrate orientation for catalysis Thermoactinomyces vulgaris
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.2.1.1 additional information
-
-
 Thermoactinomyces vulgaris
3.2.1.54 additional information
-
-
 Thermoactinomyces vulgaris

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.1 cyclodextrin + H2O cyclomaltodextrinase activity, hydrolysis of alpha-1,4-glucosidic linkages Thermoactinomyces vulgaris panose + maltoheptaose
-
 ?
3.2.1.1 isopanose + H2O cyclomaltodextrinase activity, hydrolysis of alpha-1,6-glucosidic linkages Thermoactinomyces vulgaris ?
-
 ?
3.2.1.1 additional information substrate specificity of the bifunctional enzyme, the enzyme hydrolyzes alpha-1,4-glucosidic linkages and alpha-1,6-glucosidic linkages, and performs transglycosylation reactions, overview Thermoactinomyces vulgaris ?
-
 ?
3.2.1.1 pullulan + H2O cyclomaltodextrinase activity, hydrolysis of alpha-1,4-glucosidic linkages Thermoactinomyces vulgaris panose + ?
-
 ?
3.2.1.1 starch + H2O alpha-amylase activity, preferred substrate, hydrolysis of alpha-1,4-glucosidic linkages Thermoactinomyces vulgaris maltooligosaccharides
-
 ?
3.2.1.54 cyclodextrin + H2O cyclomaltodextrinase activity, hydrolysis of alpha-1,4-glucosidic linkages Thermoactinomyces vulgaris panose + maltoheptaose
-
 ?
3.2.1.54 cyclodextrin + H2O cyclomaltodextrinase activity, hydrolysis of alpha-1,4-glucosidic linkages Thermoactinomyces vulgaris R-47 panose + maltoheptaose
-
 ?
3.2.1.54 isopanose + H2O cyclomaltodextrinase activity, hydrolysis of alpha-1,6-glucosidic linkages Thermoactinomyces vulgaris ?
-
 ?
3.2.1.54 isopanose + H2O cyclomaltodextrinase activity, hydrolysis of alpha-1,6-glucosidic linkages Thermoactinomyces vulgaris R-47 ?
-
 ?
3.2.1.54 additional information substrate specificity of the bifunctional enzyme, the enzyme hydrolyzes alpha-1,4-glucosidic linkages and alpha-1,6-glucosidic linkages, and performs transglycosylation reactions, overview Thermoactinomyces vulgaris ?
-
 ?
3.2.1.54 additional information substrate specificity of the bifunctional enzyme, the enzyme hydrolyzes alpha-1,4-glucosidic linkages and alpha-1,6-glucosidic linkages, and performs transglycosylation reactions, overview Thermoactinomyces vulgaris R-47 ?
-
 ?
3.2.1.54 pullulan + H2O cyclomaltodextrinase activity, hydrolysis of alpha-1,4-glucosidic linkages Thermoactinomyces vulgaris panose + ?
-
 ?
3.2.1.54 pullulan + H2O cyclomaltodextrinase activity, hydrolysis of alpha-1,4-glucosidic linkages Thermoactinomyces vulgaris R-47 panose + ?
-
 ?
3.2.1.54 starch + H2O alpha-amylase activity, preferred substrate, hydrolysis of alpha-1,4-glucosidic linkages Thermoactinomyces vulgaris maltooligosaccharides
-
 ?
3.2.1.54 starch + H2O alpha-amylase activity, preferred substrate, hydrolysis of alpha-1,4-glucosidic linkages Thermoactinomyces vulgaris R-47 maltooligosaccharides
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.2.1.1 alpha-amylase II
-
 Thermoactinomyces vulgaris
3.2.1.1 More cf. EC 3.2.1.54, cyclomaltodextrinase Thermoactinomyces vulgaris
3.2.1.1 TVA II
-
 Thermoactinomyces vulgaris
3.2.1.54 alpha-amylase II
-
 Thermoactinomyces vulgaris
3.2.1.54 More cf. EC 3.2.1.1, alpha-amylase Thermoactinomyces vulgaris
3.2.1.54 TVA II
-
 Thermoactinomyces vulgaris

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.1 4.8
-
 pullulan mutant Y374A Thermoactinomyces vulgaris
3.2.1.1 9.4
-
 starch mutant Y374A Thermoactinomyces vulgaris
3.2.1.1 120
-
 starch wild-type enzyme Thermoactinomyces vulgaris
3.2.1.1 140
-
 pullulan wild-type enzyme Thermoactinomyces vulgaris
3.2.1.54 4.8
-
 pullulan mutant Y374A Thermoactinomyces vulgaris
3.2.1.54 9.4
-
 starch mutant Y374A Thermoactinomyces vulgaris
3.2.1.54 120
-
 starch wild-type enzyme Thermoactinomyces vulgaris
3.2.1.54 140
-
 pullulan wild-type enzyme Thermoactinomyces vulgaris