Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975

The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus sal ivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 deg rees C, indicating that Asp-312 present in the 'sucrose box' was not the nu cleophilic Asp residue responsible for the formation of a covalent fructosy l-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp312 w as most likely an essential amino acid involved in determining acceptor rec ognition and/or stabilizing a beta-turn in the protein. In contrast, when t he 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 resid ue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Compa rison 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 cova lent fructosyl intermediate during the catalysis of sucrose by the Ftf of S . salivarius.