Involvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilization.
Multiple alignment of deduced amino-acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well-conserved catalytic domain. A portion of this domain displays homology to members of the alpha-amylase family (glycoside hydrolase family 13), which all have a (beta/alpha)8 barrel structure. In the glucansucrases, however, the alpha-helix and beta-strand elements are circularly permuted with respect to the order in family 13. Previous work has shown that amino-acid residues contributing to the active site of glucansucrases are situated in structural elements that align with those of family 13. In alpha-amylase and cyclodextrin glucanotransferase, a histidine residue has been identified that acts to stabilize the transition state, and a histidine is conserved at the corresponding position in all other members of family 13. In all the glucansucrases, however, the aligned position is occupied by glutamine. Mutants of glucosyltransferase I were constructed in which this glutamine, Gln937, was changed to histidine, glutamic acid, aspartic acid, asparagine or alanine. The effects on specific activity, ability to form glucan and ability to transfer glucose to a maltose acceptor were examined. Only histidine could substitute for glutamine and maintain Michaelis-Menten kinetics, albeit at a greatly reduced kcat, showing that Gln937 plays a functionally equivalent role to the histidine in family 13. This provides additional evidence in support of the proposed alignment of the (beta/alpha)8 barrel structures. Mutation at position 937 altered the acceptor reaction with maltose, and resulted in the synthesis of novel gluco-oligosaccharides in which alpha1,3-linked glucosyl units are joined sequentially to maltose.
Monchois
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V
Department of Oral Biology, The Dental School, University of Newcastle upon Tyne, UK.
Vignon
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Escalier
P C
PC
Svensson
B
B
Russell
R R
RR
D004795:Enzyme Stability; D005936:Glucans / Q000096:biosynthesis; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D013291:Streptococcus / Q000201:enzymology*; D013329:Structure-Activity Relationship
D005936:Glucans; D009844:Oligosaccharides; D005964:Glucosyltransferases; C020025:dextransucrase