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Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase

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Author: Kralj, S. · Geel-Schutten, G.H. van · Maarel, M.J.E.C. van der · Dijkhuizen, L.
Institution: TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO
Source:Microbiology, 7, 150, 2099-2112
Identifier: 237859
Keywords: Nutrition · Food technology · Amino acid · Asparagine · Aspartic acid · Glucan · Glucoside · Glutamic acid · Glutamine · Glycosyltransferase · Oligosaccharide · Reuteransucrase · Sucrase · Sucrose · Unclassified drug · Amino terminal sequence · Bioassay · Carbohydrate synthesis · Carboxy terminal sequence · Catalysis · Controlled study · Deletion mutant · Enzyme activity · Enzyme analysis · Enzyme inactivation · Enzyme kinetics · Lactobacillus reuteri · Leuconostoc · Molecular size · Nonhuman · Priority journal · Protein binding · Sequence alignment · Streptococcus · Amino Acid Sequence · Binding Sites · Gene Deletion · Glucans · Glycosyltransferases · Kinetics · Lactobacillus · Maltose · Molecular Sequence Data · Mutagenesis, Site-Directed · Sucrose · Lactobacillus · Lactobacillus reuteri · Leuconostoc · Streptococcus


Lactobacillus reuteri strain 121 uses sucrose for synthesis of a unique, soluble glucan ('reuteran') with mainly α-(1→4) glucosidic linkages. The gene (gtfA) encoding this glucansucrase enzyme had previously been characterized. Here, a detailed biochemical and molecular analysis of the GTFA enzyme is presented. This is believed to be the first report describing reuteransucrase enzyme kinetics and the oligosaccharides synthesized with various acceptors. Alignments of the GTFA sequence with glucansucrases from Streptococcus and Leuconostoc identified conserved amino-acid residues in the catalytic core critical for enzyme activity. Mutants Asp1024Asn, Glu1061Gln and Asp1133Asn displayed 300- to 1000-fold-reduced specific activities. To investigate the role of the relatively large N-terminal variable domain (702 amino acids) and the relatively short C-terminal putative glucan-binding domain (267 amino acids, with 11 YG repeats), various truncated derivatives of GTFA (1781 amino acids) were constructed and characterized. Deletion of the complete N-terminal variable domain of GTFA (GTFA-ΔN) had little effect on reuteran characteristics (size, distribution of glycosidic linkages), but the initial transferase activity of the mutant enzyme increased drastically. Sequential C-terminal deletions (up to six YG repeats) in GTFA-ΔN also had little effect on reuteran characteristics. However, enzyme kinetics drastically changed. Deletion of 7, 8 or 11 YG repeats resulted in dramatic loss of total enzyme activity (43-, 63- and 1000-fold-reduced specific activities, respectively). Characterization of sequential C-terminal deletion mutants of GTFA-ΔN revealed that the C-terminal domain of reuteransucrase has an important role in glucan binding. © 2004 SGM.