Title
Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase
Author
Kralj, S.
van Geel-Schutten, G.H.
van der Maarel, M.J.E.C.
Dijkhuizen, L.
TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO
Publication year
2004
Abstract
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.
Subject
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
To reference this document use:
http://resolver.tudelft.nl/uuid:f09e10f2-782a-432e-acae-ae57afdb20d9
TNO identifier
237859
ISSN
1350-0872
Source
Microbiology, 150 (7), 2099-2112
Document type
article