Mutagenesis of Asp-569 of glucosyltransferase I glucansucrase modulates glucan and oligosaccharide synthesis

Glucansucrases of oral streptococci and Legconostoc mesenteroides are enzym es of medical and biotechnological interest that synthesize ar-glucans. The y can also synthesize oligosaccharides in the presence of a sugar acceptor. Previous reports have identified an amino acid residue that may affect the structure of the glucan product; therefore, random mutagenesis of the corr esponding Asp-569 of Streptacoccus downei glucosyltransferase I (GTF-I) was used to further understanding of its involvement in the catalytic mechanis m and to evaluate how different amino acids can modulate glucan and oligosa ccharide synthesis. GTF-I variants were obtained where Asp-569 was replaced by each of the different possible classes of amino acids. These were expre ssed in Escherichia coli and purified by means of a His, tag. The results s howed that the amino acid in position 569 influences the structure of the g lucan and the size of the oligosaccharides produced by GTF-I. The results s uggest that the amino acid occupying this position is more likely to intera ct with the acceptor molecules (oligosaccharides or elongating glucan chain ) than to be directly involved in glucosyl transfer from sucrose. Engineeri ng of the equivalent position in glucansucrases thus appears to be a good t arget to expand the range of oligosaccharides synthesized.