Title
Single amino acid residue changes in subsite - 1 of inulosucrase from Lactobacillus reuteri 121 strongly influence the size of products synthesized
Author
TNO Kwaliteit van Leven TNO Voeding
Ozimek, L.K.
Kralj, S.
Kaper, T.
van der Maarel, M.J.E.C.
Dijkhuizen, L.
Publication year
2006
Abstract
Bacterial fructansucrase enzymes belong to glycoside hydrolase family 68 and catalyze transglycosylation reactions with sucrose, resulting in the synthesis of fructooligosaccharides and/or a fructan polymer. Significant differences in fructansucrase enzyme product specificities can be observed, i.e. in the type of polymer (levan or inulin) synthesized, and in the ratio of polymer versus fructooligosaccharide synthesis. The Lactobacillus reuteri 121 inulosucrase enzyme produces a diverse range of fructooligosaccharide molecules and a minor amount of inulin polymer [with beta(2-1) linkages]. The three-dimensional structure of levansucrase (SacB) of Bacillus subtilis revealed eight amino acid residues interacting with sucrose. Sequence alignments showed that six of these eight amino acid residues, including the catalytic triad (D272, E523 and D424, inulosucrase numbering), are completely conserved in glycoside hydrolase family 68. The other three completely conserved residues are located at the - 1 subsite (W271, W340 and R423). Our aim was to investigate the roles of these conserved amino acid residues in inulosucrase mutant proteins with regard to activity and product profile. Inulosucrase mutants W340N and R423H were virtually inactive, confirming the essential role of these residues in the inulosucrase active site. Inulosucrase mutants R423K and W271N were less strongly affected in activity, and displayed an altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer. Our data show that the - 1 subsite is not only important for substrate recognition and catalysis, but also plays an important role in determining the size of the products synthesized. © 2006 The Authors.
Subject
Food technology
Inulosucrase
Lactobacillus reuteri
Processivity
Site-directed mutagenesis
Amino acid
Arginine
Aspartic acid
Fructan
Fructansucrase
Fructose oligosaccharide
Glutamic acid
Glycosidase
Histidine
Inulin
Inulosucrase
Levan
Lysine
Mutant protein
Oligosaccharide
Polymer
Sucrase
Sucrose
Tryptophan
Unclassified drug
Amino acid sequence
Carbohydrate synthesis
Catalysis
Concentration (parameters)
Controlled study
Enzyme active site
Enzyme activity
Enzyme specificity
Enzyme structure
Enzyme substrate
Glycosylation
Molecular recognition
Molecular size
Nonhuman
Nucleotide sequence
Protein family
Amino Acid Substitution
Binding Sites
Hexosyltransferases
Lactobacillus reuteri
Molecular Weight
Oligosaccharides
Bacillus subtilis
Bacteria (microorganisms)
Lactobacillus reuteri
To reference this document use:
http://resolver.tudelft.nl/uuid:5731e5cd-b00c-49ca-8e1e-c87af8ec06b7
DOI
https://doi.org/10.1111/j.1742-4658.2006.05411.x
TNO identifier
239436
ISSN
1742-464X
Source
FEBS Journal, 273 (273), 4104-4113
Document type
article