Title
Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121
Author
TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO
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
Abstract
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.
Subject
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
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http://resolver.tudelft.nl/uuid:112a4a6c-e193-46c7-83d7-6d5a2298224d
DOI
https://doi.org/10.1016/s0014-5793(04)00085-7
TNO identifier
237623
ISSN
0014-5793
Source
FEBS Letters, 560 (560), 131-133
Document type
article