Replacement of the catalytic nucleophile aspartyl residue of dextran glucosidase by cysteine sulfinate enhances transglycosylation activity.
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.
From the Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8689, Japan.
Wataru
W
Saburi
Momoko
M
Kobayashi
Haruhide
H
Mori
Masayuki
M
Okuyama
Atsuo
A
Kimura
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D020125:Mutation, Missense; D013295:Streptococcus mutans / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D001224:Aspartic Acid; D005959:Glucosidases; D003545:Cysteine; C013461:cysteine sulfinic acid
Catalytic Nucleophile; Cysteine Sulfinate; Dextran Glucosidase; Enzyme Catalysis; Enzyme Kinetics; Glycoside Hydrolase Family 13; Glycoside Hydrolases; Protein Engineering; Site-specific Mutagenesis; Transglycosylation