Replacement of the Catalytic Nucleophile Aspartyl Residue of Dextran Glucosidase by Cysteine Sulfinate Enhances Transglycosylation Activity
| Autor: | Wataru Saburi, Haruhide Mori, Momoko Kobayashi, Masayuki Okuyama, Atsuo Kimura |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Transglycosylation
Glycosylation Glycoside Hydrolases Stereochemistry Mutation Missense Site-specific Mutagenesis Glycoside Hydrolase Family 13 Protein Engineering Biochemistry Dextran Glucosidase Catalysis Enzyme catalysis Streptococcus mutans Hydrolysis Nucleophile Bacterial Proteins Catalytic Nucleophile Organic chemistry Glycoside hydrolase Enzyme kinetics Cysteine Molecular Biology Enzyme Kinetics chemistry.chemical_classification Aspartic Acid Wild type Cysteine Sulfinate Cell Biology Enzyme chemistry Amino Acid Substitution Enzyme Catalysis Enzymology Glucosidases |
| Popis: | 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. Background: A mutant dextran glucosidase where the nucleophilic Asp (-COO−) was replaced by cysteine sulfinate (-SOO−) was characterized. Results: The cysteine sulfinate-introduced enzyme showed higher transglucosylation activity than wild type enzyme. Conclusion: The more acidic nucleophile (-SOO−) increased transglycosylation activity. Significance: A novel strategy to enhance transglycosylation by glycosidases is proposed. |
| Databáze: | OpenAIRE |
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