Mutagenesis of asp-569 of glucosyltransferase I glucansucrase modulates glucan and oligosaccharide synthesis.
Glucansucrases of oral streptococci and Leuconostoc mesenteroides are enzymes of medical and biotechnological interest that synthesize alpha-glucans. They 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 corresponding Asp-569 of Streptococcus downei glucosyltransferase I (GTF-I) was used to further understanding of its involvement in the catalytic mechanism and to evaluate how different amino acids can modulate glucan and oligosaccharide synthesis. GTF-I variants were obtained where Asp-569 was replaced by each of the different possible classes of amino acids. These were expressed in Escherichia coli and purified by means of a His(6) tag. The results showed that the amino acid in position 569 influences the structure of the glucan and the size of the oligosaccharides produced by GTF-I. The results suggest that the amino acid occupying this position is more likely to interact with the acceptor molecules (oligosaccharides or elongating glucan chain) than to be directly involved in glucosyl transfer from sucrose. Engineering of the equivalent position in glucansucrases thus appears to be a good target to expand the range of oligosaccharides synthesized.
Monchois
V
V
Department of Oral Biology, The Dental School, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom.
Vignon
M
M
Russell
R R
RR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D001426:Bacterial Proteins; D005936:Glucans / Q000096:biosynthesis*; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases; D006801:Humans; D007934:Leuconostoc / Q000201:enzymology*; D008969:Molecular Sequence Data; D009055:Mouth / Q000382:microbiology; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000096:biosynthesis*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D005936:Glucans; D009844:Oligosaccharides; D011506:Proteins; D011994:Recombinant Proteins; C067118:GTF-I protein, Streptococcus; D001224:Aspartic Acid; D016695:Glycosyltransferases; D005964:Glucosyltransferases; C489364:glucosyltransferase I; C033837:alternansucrase; C020025:dextransucrase; D006026:Glycoside Hydrolases