Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase

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.

Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

“…Enzymatic Activity Assays-The enzymatic activities of GTF180-⌬N (mutants) were measured as previously described (6). Briefly, enzyme assays were performed with 30 nM enzyme in 25 mM sodium acetate buffer, pH 4.5, at 50°C.…”

“…The cells were collected by centrifugation (10 min, 4°C, 10,000 ϫ g) and washed with 50 mM TrisHCl buffer, pH 8.0. The (mutant) glucansucrase enzymes were purified as previously described (6).…”

“…Three catalytically crucial residues of glucansucrases have been identified in previous studies (6,17,18). In glycoside hydrolase family 70 enzymes, Asp 1025 (catalytic nucleophile, GTF180 numbering) is involved in the formation of a covalent glucosyl-enzyme intermediate, Glu 1063 is the acid/base catalyst, and Asp 1136 is the transition state stabilizer (19).…”

Binding Interactions Between α-glucans from Lactobacillus reuteri and Milk Proteins Characterised by Surface Plasmon Resonance

“…Production and isolation of the α-glucans have previously been described. 7,22,23 Milk Proteins β-lactoglobulin and κ-casein were purchased from Sigma-Aldrich (St. Louis, MO, USA). β-lactoglobulin (5 gL -1 ) was heat-denatured (85ºC, 15 min) in sodium phosphate (50 mM, 30 mM NaCl, pH 6.8).…”

“…In the present study, glucans from different strains of Lactobacillus reuteri (ATCC-55730, 180, ML1 and 121) of varying molecular weight, linkage types, and degree of branching 7,[21][22][23][24] were analysed for binding to native β-lactoglobulin, denatured β-lactoglobulin and κ-casein in the range pH 4.0-5.5 by using SPR. The extent of individual interactions was correlated with the molecular properties of the glucans.…”