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Site‐directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121
Author(s) -
Ozimek L.K,
van Hijum S.A.F.T,
van Koningsveld G.A,
van der Maarel M.J.E.C,
van Geel-Schutten G.H,
Dijkhuizen L
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(04)00085-7
Subject(s) - levansucrase , lactobacillus reuteri , glycoside hydrolase , bacillus subtilis , chemistry , mutant , site directed mutagenesis , mutagenesis , biochemistry , stereochemistry , nucleophile , enzyme , catalysis , lactobacillus , biology , bacteria , fermentation , genetics , gene
Bacterial fructosyltransferases (FTFs) are retaining‐type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high‐resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935–941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site‐directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site‐directed mutants of the three putative catalytic residues of the Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5–4×10 5 times reduction of total sucrase activity.