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Single amino acid residue changes in subsite - 1 of inulosucrase from Lactobacillus reuteri 121 strongly influence the size of products synthesized

Author: Ozimek, L.K. · Kralj, S. · Kaper, T. · Maarel, M.J.E.C. van der · Dijkhuizen, L.
Institution: TNO Kwaliteit van Leven TNO Voeding
Source:FEBS Journal, 17, 273, 4104-4113
Identifier: 239436
doi: doi:10.1111/j.1742-4658.2006.05411.x
Keywords: 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


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 β(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.