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

Abstract

The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 °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 β-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.

Prediction of a common β‐propeller catalytic domain for fructosyltransferases of different origin and substrate specificity

“…The RDP motif of levansucrases from Acetobacter diazotrophicus~Batista et al, 1999! andStreptococcus salivarius~Song &Jacques, 1999! was found to be involved in Table 1, and the conserved residues are in bold.…”

“…Additionally, the sequencestructure alignment in Figure 4B showed a high correspondence between the secondary structure elements, which are characteristics of the pseudo sixfold symmetry. The equivalent residues Asp309 and Asp397 in the RDP motif of levansucrases from A. diazotrophicus and S. salivarius, respectively, were found to be implicated in binding or splitting of sucrose~Batista et al, 1999;Song & Jacques, 1999!. The RDP motif is close in space to Arg331~in the vicinity of the conserved region VII in Fig.…”

“…The most reliable structural model was obtained using the crystal structure of neuraminidase~Protein Data Bank file: 5nn9! as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases~Batista Song & Jacques, 1999!. The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern $H-x~2!-@PTV#-x~4!-@LIVMA#-@NSCAYG#-@DE#-P-@NDSC#-@GA#% for this family.…”

Kinetic properties of an inulosucrase from Lactobacillus reuteri 121

“…The fact that these levansucrase enzymes produce levan and 1‐kestose might indicate the presence of two binding sites in FTF enzymes: one site generates the 1‐kestose primer molecules, which are subsequently used by a second binding site in the polymerization process. A second binding site for the polymerization reaction has previously been proposed for the FTF enzyme of S. salivarius [21].…”

Site‐directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121

“…Residue D247 was identified as a transition state stabilizer based on the observation that it forms strong hydrogen bonds with the C3′ and C4′ hydroxyls of the fructosyl unit, but it is too far away from either the C2′ hydroxyl or the glycosidic oxygen to be one of the residues directly involved in catalysis. Additional evidence for the role of D247 comes from mutational studies on the equivalent residue in the levansucrases of Z. mobilis (D194), Streptococcus salivarius (D397) and Gluconacetobacter diazotrophicus SRT4 (D309) [7–9]. With respect to the catalytic nucleophile, no experimental mutagenesis studies have been reported yet.…”