The mechanism of dextransucrase action
Additionally, D536N dextransucrase showed complete suppression of oligosaccharide synthesis activities.
The mechanism of dextransucrase action, direction …
D533A and D536A dextransucrases showed reduced dextran synthesis activities, 2.3% and 40.8% of DSRB742 dextransucrase, respectively, and D533N, D536N, H643A, end H643N dextransucrases showed complete suppression of dextran synthesis activities altogether.
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The activation of DSR-S by maltose described previously (, ) 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 (). In the presence of maltose, the formation of a -glucosyl–enzyme complex before sugar is transferred to the acceptor should be the limiting step (). This effect is observed with deleted proteins, which supports the idea that the kinetics of -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 (), 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 (, ), this supports the hypothesis that the C-terminal portion is not involved in the process which results in oligosaccharide formation.
establish the mechanism of action of dextransucrase
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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 (, ). 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 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.
The Mechanism of Dextransucrase Action
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Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats. In order to determine the role of the C-terminal region of DSR-S in dextran or oligosaccharide synthesis, four DSR-S genes with deletions at the 3′ end were constructed. The results showed that the C-terminal region modulated the initial velocity of dextran synthesis but that the Km for sucrose, the optimum pH, and the activation energy were all unaffected by the deletions. The C-terminal domain modulated the rate of oligosaccharide synthesis whatever acceptor molecule was used (a good acceptor molecule such as maltose or a poor acceptor molecule such as fructose). The C-terminal domain seemed to play no role in the catalytic process in dextran and oligosaccharide synthesis. In fact, it seems that the role of the C-terminal domain of DSR-S may be to facilitate the translation of dextran and oligosaccharides from the catalytic site.
Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a 1,527-amino-acid glucosyltransferase (EC ) that catalyzes the synthesis from sucrose of a soluble dextran in which more than 95% of the -glucosyl units are α(1-6) linked (, ). This dextran has many important industrial and medical uses (). In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward oligosaccharide synthesis (, ).
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mechanism of dextransucrase action
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Leuconostoc mesenteroides, a common lactic acid bacterium, displays a wide range of biocatalytic properties, which are potentially useful for industrial carbohydrate modifications. The use of L. mesenteroides for the production of dextran via whole cell fermentation, for leucrose synthesis with dextransucrase, for the synthesis of alternan and gluco-oligosaccharides with alternansucrase, for mannitol fermentation with viable L. mesenteroides cells, and for the synthesis of α-D-glucose- 1 -phosphate using sucrose phosphorylase, are discussed.
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In the present paper we describe a sequence analysis of the C-terminal portion of L. mesenteroides NRRL B-512F DSR-S and the effect of sequential deletions on the biochemical properties of DSR-S. We paid particular attention to characterizing the dextran and oligosaccharide synthesis activities of mutants in order to better understand the role of the C-terminal region of DSR-S in the catalytic mechanism.
Preparative Biochemistry and Biotechnology - Taylor & …
Acceptor reaction of a highly purified dextransucrase with maltose and oligosaccharides: Application to the synthesis of controlledmolecular- weight dextrans, Carbohydr.
Preparative Biochemistry and Biotechnology ..
The binding sites for sucrose and dextran are separate sites on DSR-S (), and the catalytic site responsible for cleavage of sucrose is located in its N-terminal region (, , ). 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.
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