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Replacement of the Catalytic Nucleophile Aspartyl Residue of Dextran Glucosidase by Cysteine Sulfinate Enhances Transglycosylation Activity

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Title: Replacement of the Catalytic Nucleophile Aspartyl Residue of Dextran Glucosidase by Cysteine Sulfinate Enhances Transglycosylation Activity
Authors: Saburi, Wataru Browse this author →KAKEN DB
Kobayashi, Momoko Browse this author
Mori, Haruhide Browse this author →KAKEN DB
Okuyama, Masayuki Browse this author →KAKEN DB
Kimura, Atsuo Browse this author →KAKEN DB
Keywords: Enzyme Catalysis
Enzyme Kinetics
Glycoside Hydrolases
Site-specific Mutagenesis
Protein Engineering
Catalytic Nucleophile
Cysteine Sulfinate
Dextran Glucosidase
Glycoside Hydrolase Family 13
Transglycosylation
Issue Date: 1-Nov-2013
Publisher: American Society for Biochemistry and Molecular Biology (ASBMB)
Journal Title: Journal of Biological Chemistry
Volume: 288
Issue: 44
Start Page: 31670
End Page: 31677
Publisher DOI: 10.1074/jbc.M113.491449
PMID: 24052257
Abstract: Dextran glucosidase from Streptococcus mutans (SmDG) catalyzes the hydrolysis of an α-1,6-glucosidic linkage at the nonreducing end of isomaltooligosaccharides and dextran. This enzyme has an Asp-194 catalytic nucleophile and two catalytically unrelated Cys residues, Cys-129 and Cys-532. Cys-free SmDG was constructed by replacement with Ser (C129S/C532S (2CS), the activity of which was the same as that of the wild type, SmDG). The nucleophile mutant of 2CS was generated by substitution of Asp-194 with Cys (D194C-2CS). The hydrolytic activity of D194C-2CS was 8.1 × 10⁻⁴ % of 2CS. KI-associated oxidation of D194C-2CS increased the activity up to 0.27% of 2CS, which was 330 times higher than D194C-2CS. Peptide-mapping mass analysis of the oxidized D194C-2CS (Ox-D194C-2CS) revealed that Cys-194 was converted into cysteine sulfinate. Ox-D194C-2CS and 2CS shared the same properties (optimum pH, pI, and substrate specificity), whereas Ox-D194C-2CS had much higher transglucosylation activity than 2CS. This is the first study indicating that a more acidic nucleophile (-SOO−) enhances transglycosylation. The introduction of cysteine sulfinate as a catalytic nucleophile could be a novel approach to enhance transglycosylation.
Rights: This research was originally published in The Journal of biological chemistry. Wataru Saburi, Momoko Kobayashi, Haruhide Mori, Masayuki Okuyama, and Atsuo Kimura. "Replacement of the Catalytic Nucleophile Aspartyl Residue of Dextran Glucosidase by Cysteine Sulfinate Enhances Transglycosylation Activity". J Biol Chem. 2013; Vol288(44):pp31670-pp31677. © the American Society for Biochemistry and Molecular Biology.
Type: article
URI: http://hdl.handle.net/2115/67776
Appears in Collections:農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 佐分利 亘

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