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Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121

Author: Ozimek, L.K. · Hijum, S.A.F.T. van · Koningsveld, G.A. van · Maarel, M.J.E.C. van der · Geel-Schutten, G.H. van · Dijkhuizen, L.
Institution: TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO
Source:FEBS Letters, 1-3, 560, 131-133
Identifier: 237623
doi: doi:10.1016/S0014-5793(04)00085-7
Keywords: Food technology · Catalytic residue · Fructosyltransferase · Inulosucrase · Levansucrase · Mutagenesis · Acid · Base · Glycosidase · Levansucrase · Mutant protein · Stabilizing agent · Sucrase · Transferase · Bacterium mutant · Enzyme activity · Nonhuman · Site directed mutagenesis · Amino Acid Motifs · Amino Acid Sequence · Amino Acid Substitution · Catalysis · Circular Dichroism · Cloning, Molecular · Conserved Sequence · Escherichia coli · Gene Expression · Genes, Bacterial · Hexosyltransferases · Kinetics · Lactobacillus · Molecular Sequence Data · Mutagenesis, Site-Directed · Sequence Homology, Amino Acid · Substrate Specificity · Bacillus subtilis · Bacteria (microorganisms) · Lactobacillus reuteri


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 1.5-4×105 times reduction of total sucrase activity. © 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.