Molecular characterization of a novel glucosyltransferase from Lactobacillus reuteri strain 121 synthesizing a unique, highly branched glucan with alpha-(1-->4) and alpha-(1-->6) glucosidic bonds.
Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the alpha-(1-->4) glucosidic type. The glucan also contains alpha-(1-->6)-linked glucosyl units and 4,6-disubstituted alpha-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M(r) of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.
Centre for Carbohydrate Bioengineering, TNORUG, Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
S
S
Kralj
G H
GH
van Geel-Schutten
H
H
Rahaoui
R J
RJ
Leer
E J
EJ
Faber
M J E C
MJ
van der Maarel
L
L
Dijkhuizen
D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D005936:Glucans; D005964:Glucosyltransferases