Rational transformation of Lactobacillus reuteri 121 reuteransucrase into a dextransucrase.
Glucansucrase or glucosyltransferase (GTF) enzymes of lactic acid bacteria display high sequence similarity but catalyze synthesis of different alpha-glucans (e.g., dextran, mutan, alternan, and reuteran) from sucrose. The variations in glucosidic linkage specificity observed in products of different glucansucrase enzymes appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. This notion was derived from mutagenesis of amino acids of GTFA (reuteransucrase) from Lactobacillus reuteri strain 121 putatively involved in acceptor substrate binding. A triple amino acid mutation (N1134S:N1135E:S1136V) in a region immediately next to the catalytic Asp1133 (putative transition state stabilizing residue) converted GTFA from a mainly alpha-(1-->4) ( approximately 45%, reuteran) to a mainly alpha-(1-->6) ( approximately 80%, dextran) synthesizing enzyme. The subsequent introduction of mutation P1026V:I1029V, involving two residues located in a region next to the catalytic Asp1024 (nucleophile), resulted in synthesis of an alpha-glucan containing only a very small percentage of alpha-(1-->4) glucosidic linkages ( approximately 5%) and a further increased percentage of alpha-(1-->6) glucosidic linkages ( approximately 85%). This changed glucosidic linkage specificity was also observed in the oligosaccharide products synthesized by the different mutant GTFA enzymes from (iso)maltose and sucrose. Amino acids crucial for glucosidic linkage type specificity of reuteransucrase have been identified in this report. The data show that a combination of mutations in different regions of GTF enzymes influences the nature of both the glucan and oligosaccharide products. The amino acids involved most likely contribute to sugar-binding acceptor subsites in glucansucrase enzymes.
Kralj
Slavko
S
Centre for Carbohydrate Bioengineering, TNO-University of Groningen, Haren, The Netherlands.
van Geel-Schutten
Ineke G H
IG
Faber
Elly J
EJ
van der Maarel
Marc J E C
MJ
Dijkhuizen
Lubbert
L
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005936:Glucans / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D007534:Isomaltose / Q000378:metabolism; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering; D016415:Sequence Alignment; D013057:Spectrum Analysis; D013393:Sucrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism
D001426:Bacterial Proteins; D005936:Glucans; D013395:Sucrose; D007534:Isomaltose; D008320:Maltose; D005964:Glucosyltransferases; C020025:dextransucrase; D013393:Sucrase; D005947:Glucose