Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I.
Related streptococcal and Leuconostoc mesenteroides glucansucrases are enzymes of medical and biotechnological interest. Molecular modelling has suggested that the catalytic domain contains a circularly permuted version of the (beta/alpha)8 barrel structure found in the amylase superfamily, and site-directed mutagenesis has identified critical amino acids in this region. In this study, sequential N-terminal truncations of Streptococcus downei GTF-I showed that key amino acids are also present in the first one-third of the core domain. Mutations were introduced at Trp-344, Glu-349 and His-355, residues that are conserved in all glucansucrases and lie within a region which is a target for inhibitory antibodies. W344L, E349L and H355V substitutions were assayed for their effect on mutan synthesis and also on oligosaccharide synthesis with various acceptors. It appeared that Trp-344 and His-355 are involved in the action mechanism of GTF-I; His-355 may also play a role in a binding subsite necessary for oligosaccharide and glucan elongation.
Monchois
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V
Department of Oral Biology, Dental School, University of Newcastle upon Tyne, UK. vincent.monchois@ncl.ac.uk
Vignon
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M
Russell
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RR
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005936:Glucans / Q000096:biosynthesis; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D016695:Glycosyltransferases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017384:Sequence Deletion; D013291:Streptococcus / Q000201:enzymology*
D005936:Glucans; D009841:Oligonucleotides; D011994:Recombinant Proteins; D016695:Glycosyltransferases; D005964:Glucosyltransferases; C033837:alternansucrase; D006026:Glycoside Hydrolases