Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase.
Lactobacillus reuteri strain 121 uses sucrose for synthesis of a unique, soluble glucan ('reuteran') with mainly alpha-(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-Delta N) 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-Delta N 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-Delta N revealed that the C-terminal domain of reuteransucrase has an important role in glucan binding.
Kralj
S
S
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands.|Centre for Carbohydrate Bioengineering (CCB), TNO-University of Groningen, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
van Geel-Schutten
G H
GH
Centre for Carbohydrate Bioengineering (CCB), TNO-University of Groningen, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.|Innovative Ingredients and Products Department, TNO-Nutrition and Food Research, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
van der Maarel
M J E C
MJEC
Innovative Ingredients and Products Department, TNO-Nutrition and Food Research, Rouaanstraat 27, 9723 CC, Groningen, The Netherlands.|Centre for Carbohydrate Bioengineering (CCB), TNO-University of Groningen, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
Dijkhuizen
L
L
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands.|Centre for Carbohydrate Bioengineering (CCB), TNO-University of Groningen, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
D000595:Amino Acid Sequence; D001665:Binding Sites; D017353:Gene Deletion; D005936:Glucans / Q000378:metabolism*; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013395:Sucrose / Q000378:metabolism
D005936:Glucans; D013395:Sucrose; D008320:Maltose; D016695:Glycosyltransferases; C033837:alternansucrase