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It is shown that exchanges of single invariant amino acids in two C-terminal catalytic domain segments of the glucosyltransferase R (GtfR) strongly affect its catalytic properties. Drastic decreases of activity through re- or displacements of Tyr965 demonstrate a crucial role of this residue. Similarly, exchanges of amino acids Asp1004, Val1006, and Tyr1011 profoundly influenced catalytic parameters. These results are interpreted on the basis of a homology model of the catalytic domain. They are consistent with the view that Tyr965 is a constituent of the substrate-binding pocket and directly contacts the sucrose molecule, whereas the other critical residues contribute to the required positioning of Tyr965 and other active site residues.

Segments that may crucially influence the catalytic behaviour of glucosyltransferases of the glucansucrase type were selected for modification. This was done by sequence alignments, followed by structural modelling of the putative catalytic domain, based on a permuted form of the glucosyltransferase R (GtfR) of Streptococcus oralis. Five selected regions, located in the C-terminal half of the potential catalytic domain, were replaced by segments found at equivalent positions in other glucosyltransferases. The exchanges of four of these regions significantly affected catalysis by GtfR. This identified C-terminal determinants for substrate binding and turnover and supports the so-called permutation hypothesis with respect to enzymes of the glucansucrase type. Based on the model, roles are proposed for specific residues. Major effects appear to involve a re-positioning of the C-terminal Tyr965 that very likely serves as a hydrophobic platform for the substrate.