Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975.
The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 degrees C, indicating that Asp-312 present in the 'sucrose box' was not the nucleophilic Asp residue responsible for the formation of a covalent fructosyl-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp-312 was most likely an essential amino acid involved in determining acceptor recognition and/or stabilizing a beta-turn in the protein. In contrast, when the Asp-397 of the Ftf present in the conserved triplet RDP motif of all 60 bacterial and plant family-32 glycosylhydrolases was mutated to a Ser residue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Comparison of published data from other site-directed mutated enzymes implicated the Asp residue in the RDP motif as the one that may form a transient covalent fructosyl intermediate during the catalysis of sucrose by the Ftf of S. salivarius.
Song
D D
DD
Institute of Dental Research, 2 Chalmers Street, Surry Hills, NSW 2010, Australia.
Jacques
N A
NA
D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D001426:Bacterial Proteins
D001224:Aspartic Acid; D017931:DNA Primers; D006602:Hexosyltransferases; D011994:Recombinant Proteins; D014364:Tryptophan; C021451:inulosucrase; D001426:Bacterial Proteins