Like all GTFs whose sequences are known (9
), the C-terminal domain of DSR-S from L. mesenteroides
NRRL B-512F is composed of a series of repeats homologous to A and C repeats. The terminal portion contains repeat motifs which are not highly conserved but possess the main characteristics of the YG repeats (9
) that we call N repeats. This structure has not been identified in streptoccocal GTFs. In order to gain further insight into the role of C-terminal repeats in GTF activity, the effects of engineered deletions on both dextran and oligosaccharide synthesis were examined.
As with streptococcal GTFs (1
), the C-terminal portion of DSR-S is crucial for maintaining a high initial rate of consumption of sucrose and a high initial rate of synthesis of dextran. There is a direct correlation between dextran synthesis and sucrose consumption, which indicates that deletions have no effect on the ratio of sucrose hydrolysis to polymer synthesis. This suggests that the C-terminal domain of DSR-S does not facilitate the transfer of glucosyl residues on the dextran chain.
The binding sites for sucrose and dextran are separate sites on DSR-S (15
), and the catalytic site responsible for cleavage of sucrose is located in its N-terminal region (19
). The fact that deletions do not have a drastic effect on the Km
for sucrose suggests that they do not alter the ability of DSR-S to bind the substrate sucrose. Moreover, the activation energy of the dextran synthesis reaction is not affected by deletions, which shows that the energy level of the transition state is not modified. The optimum pH does not change. The distribution of local charges in the catalytic site of DSR-S and the distribution of these charges in the truncated enzymes are the same, which indicates that the sucrose binding site is not directly affected by deletions.
The reaction velocity is the only parameter which is strongly influenced by deletions; deletions in the C terminus of DSR-S result in decreases in the initial reaction rate. Although the truncated proteins seem to be more sensitive to thermal denaturation than DSR-S is, this difference cannot explain the decreases in the initial rate observed with the truncated enzymes. The presence of the three first repeats is sufficient to maintain a detectable dextran synthesis activity, but the last 85 amino acid residues are particularly crucial for activity. However, without additional evidence it is not possible to say whether the size of the C-terminal portion of DSR-S alone is crucial for maintaining activity or whether there is a direct correlation between the absence of a nontypical N series of repeats in the C-terminal domain and the decrease in activity.
In the case of the dextran synthesis reaction, it has been proposed that translation of the growing dextran is the limiting step in the reaction, perhaps because of steric hindrance (7
). Like the initial velocity of the reaction, the glucan-binding properties of the C-terminal domain are also altered by deletions; the activator effect of dextran T70 does not occur with DSR-S3. Thus, because of its glucan-binding properties, the C-terminal domain of DSR-S could have a positive effect on the reaction velocity by making translation of the growing dextran from the catalytic site easier.
The effect of deletions on oligosaccharide synthesis has not been examined previously, but such a study could provide interesting information because mechanisms of synthesis are different; transfer of glucosyl residues occurs at the nonreducing ends of oligosaccharides, while synthesis of polymers occurs at the reducing ends (27
). Maltose and fructose were used as examples of good and bad acceptors, respectively. In both cases, the velocity of oligosaccharide synthesis was also dramatically affected by deletions. As in the dextran synthesis reaction, the C-terminal portion of DSR-S is crucial for maintaining a high initial rate of oligosaccharide production.
The activation of DSR-S by maltose described previously (24
) is even more pronounced with truncated enzymes. Paul et al. interpreted this effect as the result of a change in a limiting step of the reaction (24
). In the presence of maltose, the formation of a d
-glucosyl–enzyme complex before sugar is transferred to the acceptor should be the limiting step (24
). This effect is observed with deleted proteins, which supports the idea that the kinetics of d
-glucosyl–enzyme complex formation in the presence of maltose is not modified by deletions. The overall yields of oligosaccharides are equivalent in the presence of maltose. As previously described for dextransucrase produced by L. mesenteroides
NRRL B-512F (29
), only the ratio of sucrose concentration to maltose concentration had an effect on these yields. According to the proposed mechanisms for the acceptor reaction with maltose (28
), this supports the hypothesis that the C-terminal portion is not involved in the process which results in oligosaccharide formation.
However, the distributions of the products are not similar. A 170-amino-acid deletion in the C-terminal domain of DSR-S results in an increase in the percentage of the longest oligosaccharides produced (OD6). When the C-terminal domain is truncated, the oligosaccharides may stay longer in the microenvironment of the catalytic site, which allows the oligosaccharide chain to elongate. Thus, it seems that the role of the C-terminal domain of DSR-S in oligosaccharide synthesis is to facilitate removal of the oligosaccharides from the catalytic site. In this case, the C-terminal glucan-binding domain of DSR-S also appears to be an oligosaccharide-binding domain.
As previously described (5
), the presence of the poor acceptor fructose decreases the reaction rate of DSR-S. Deletions tend to suppress this inhibitory effect, and fructose is a strong acceptor with DSR-S3; both the reaction rate and the yield increase in its presence. Böker et al. (5
) have proposed that the leucrose synthesis reaction is slower than the dextran synthesis reaction but inhibits dextran chain elongation. With DSR-S3, the leucrose synthesis reaction may be faster than the dextran synthesis reaction. Thus, the reaction velocity would not be limited by dextran elongation but would be limited by the step that occurs in the acceptor reaction in the presence of a strong acceptor, such as maltose.