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

Saburi, Wataru; Kobayashi, Momoko; Mori, Haruhide; Okuyama, Masayuki; Kimura, Atsuo

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

Saburi, Wataru; Kobayashi, Momoko; Mori, Haruhide et al.

Journal of Biological Chemistry, 2013, 288(44), 31670-31677

Permalink : https://hdl.handle.net/2115/67776

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アクセス権	open access
URI	https://hdl.handle.net/2115/67776
タイトル	Replacement of the Catalytic Nucleophile Aspartyl Residue of Dextran Glucosidase by Cysteine Sulfinate Enhances Transglycosylation Activity
著者	Saburi, Wataru ; Kobayashi, Momoko ; Mori, Haruhide ; Okuyama, Masayuki ; Kimura, Atsuo
e-Rad_Researcher 00598089 著者名 Saburi, Wataru
著者名 Kobayashi, Momoko
e-Rad_Researcher 80241363 著者名 Mori, Haruhide
e-Rad_Researcher 00344490 著者名 Okuyama, Masayuki
e-Rad_Researcher 90186312 著者名 Kimura, Atsuo
言語	英語
キーワード	Enzyme Catalysis
	Enzyme Kinetics
	Glycoside Hydrolases
	Site-specific Mutagenesis
	Protein Engineering
	Catalytic Nucleophile
	Cysteine Sulfinate
	Dextran Glucosidase
	Glycoside Hydrolase Family 13
	Transglycosylation
発行日	2013-11-01
出版者	American Society for Biochemistry and Molecular Biology (ASBMB)
収録物名	Journal of Biological Chemistry
巻(号)	288(44)
ページ	31670-31677
抄録	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.
資源タイプ	学術雑誌論文
出版タイプ	Version of Record
権利情報	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.
PISSN	0021-9258
EISSN	1083-351X
NCID	AA00251083
関連情報	PMID 24052257
関連情報 (isIdenticalTo)	出版社版DOI https://doi.org/10.1074/jbc.M113.491449