Complex Structures of Thermoactinomyces vulgaris R-47 α-Amylase 1 with Malto-oligosaccharides Demonstrate the Role of Domain N Acting as a Starch-binding Domain

Abe, Akemi; Tonozuka, Takashi; Sakano, Yuji; Kamitori, Shigehiro

doi:10.1016/j.jmb.2003.10.078

Cited by 93 publications

(97 citation statements)

References 39 publications

(36 reference statements)

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“…Interactions between acarbose and the enzyme found in this study are very similar to those in other ␣-amylase-acarbose complex structures (25)(26)(27)(28)(29)(30).…”

Section: Resultssupporting

confidence: 86%

“…density strongly showed a binding oligosaccharide composed of five saccharide units, Glc-␣-(1,4)-Glc-␣-(1,4)-cyclitol-␣-(1,4)-6-deoxyglucose-␣-(1,4)-Glc. This is because TVAII has activity for both hydrolysis and transglycosylation, and the transglycosylation of acarbose is frequently found in acarbose complexes of the glycoside hydrolase family 13 (25)(26)(27)(28)(29)(30). For discussion, saccharide units of acarbose are designated Glc Ϫ3 , Glc Ϫ2 , Cyt Ϫ1 , Glc ϩ1 , and Glc ϩ2 from the nonreducing to reducing end.…”

Section: Resultsmentioning

confidence: 99%

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Complex Structures of Thermoactinomyces vulgaris R-47 α-Amylase 2 with Acarbose and Cyclodextrins Demonstrate the Multiple Substrate Recognition Mechanism

Ohtaki

Mizuno

Tonozuka

et al. 2004

Journal of Biological Chemistry

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Thermoactinomyces vulgaris R-47 ␣-amylase 2 (TVAII) has the unique ability to hydrolyze cyclodextrins (CDs), with various sized cavities, as well as starch. To understand the relationship between structure and substrate specificity, x-ray structures of a TVAII-acarbose complex and inactive mutant TVAII (D325N/D421N)/␣-, ␤-and ␥-CDs complexes were determined at resolutions of 2.9, 2.9, 2.8, and 3.1 Å, respectively. In all complexes, the interactions between ligands and enzymes at subsites ؊1, ؊2, and ؊3 were almost the same, but striking differences in the catalytic site structure were found at subsites ؉1 and ؉2, where Trp 356 and Tyr 374 changed the conformation of the side chain depending on the structure and size of the ligands. Trp 356 and Tyr 374 are thought to be responsible for the multiple substraterecognition mechanism of TVAII, providing the unique substrate specificity. In the ␤-CD complex, the ␤-CD maintains a regular conical structure, making it difficult for Glu 354 to protonate the O-4 atom at the hydrolyzing site as a previously proposed hydrolyzing mechanism of ␣-amylase. From the x-ray structures, it is suggested that the protonation of the O-4 atom is possibly carried out via a hydrogen atom of the inter-glucose hydrogen bond at the hydrolyzing site.

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“…Interactions between acarbose and the enzyme found in this study are very similar to those in other ␣-amylase-acarbose complex structures (25)(26)(27)(28)(29)(30).…”

Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Complex Structures of Thermoactinomyces vulgaris R-47 α-Amylase 2 with Acarbose and Cyclodextrins Demonstrate the Multiple Substrate Recognition Mechanism

Ohtaki

Mizuno

Tonozuka

et al. 2004

Journal of Biological Chemistry

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“…Indeed, the structure and biochemistry of several family 20 CBMs, which bind to starch, have been analysed extensively (see [6][7][8][9][10][11][12][13] for examples). Furthermore, numerous crystal structures of starch-modifying enzymes have revealed malto-oligosaccharide-binding sites that are distinct from the substrate-binding cleft, indicating that these enzymes also contain starch-binding CBMs [14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

Boraston

Bolam

Gilbert

et al. 2004

Biochemical Journal

1,779

1,724

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The enzymic degradation of insoluble polysaccharides is one of the most important reactions on earth. Despite this, glycoside hydrolases attack such polysaccharides relatively inefficiently as their target glycosidic bonds are often inaccessible to the active site of the appropriate enzymes. In order to overcome these problems, many of the glycoside hydrolases that utilize insoluble substrates are modular, comprising catalytic modules appended to one or more non-catalytic CBMs (carbohydrate-binding modules). CBMs promote the association of the enzyme with the substrate. In view of the central role that CBMs play in the enzymic hydrolysis of plant structural and storage polysaccharides, the ligand specificity displayed by these protein modules and the mechanism by which they recognize their target carbohydrates have received considerable attention since their discovery almost 20 years ago. In the last few years, CBM research has harnessed structural, functional and bioinformatic approaches to elucidate the molecular determinants that drive CBM-carbohydrate recognition. The present review summarizes the impact structural biology has had on our understanding of the mechanisms by which CBMs bind to their target ligands.

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“…This domain is usually localized at the C-terminal end 393 of enzymes (Svensson et al 1989). A few exceptions are the Rhizopus oryzae glucoamylase (Ashikari et al 1986;Takahashi et al 1985), the Thermoactinomyces vulgaris "α-amylase" (Abe et al 2004) and the Thermotoga maritima pullulanase (Bibel et al 1998), which present their SBDs at the N-terminus. Production of α-amylase byB.…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

et al. 2010

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Raw starch is the most abundant source of glucose in the world. Therefore, finding enzymes capable of digesting raw starch would find high industrial demand. The α-amylase gene of Bacillus amyloliquefaciens ATCC 23842 was amplified, cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme was purified to apparent homogeneity using ion exchange and gel filtration chromatography. The raw-starch digestibility of the purified enzyme was characterized by studying the hydrolysis and adsorption rate on a variety of raw starches (potato, cassava, corn, wheat and rice). The raw-starch digestion was further confirmed by scanning electron microscopy studies, which revealed an effective rate of hydrolysis. The kinetic studies revealed a relatively low Km of 2.76 mg/mL, exhibiting high affinity towards the soluble starch as the most preferred substrate and the inhibition kinetic studies revealed a high Ki value (350 mM).

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Complex Structures of Thermoactinomyces vulgaris R-47 α-Amylase 1 with Malto-oligosaccharides Demonstrate the Role of Domain N Acting as a Starch-binding Domain

Cited by 93 publications

References 39 publications

Complex Structures of Thermoactinomyces vulgaris R-47 α-Amylase 2 with Acarbose and Cyclodextrins Demonstrate the Multiple Substrate Recognition Mechanism

Complex Structures of Thermoactinomyces vulgaris R-47 α-Amylase 2 with Acarbose and Cyclodextrins Demonstrate the Multiple Substrate Recognition Mechanism

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

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