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Dextransucrase and the mechanism for dextran biosynthesis.
John F Robyt1, Seung-Heon Yoon, Rupendra Mukerjea
1Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA. jrobyt@iastate.edu
Carbohydrate Research
|October 17, 2008
Summary
Dextransucrase enzyme uses sucrose to synthesize dextran, not glucose or sucrose as primers. Glucose from sucrose is added to the growing dextran chain
Area of Science:
- Enzymology
- Carbohydrate Chemistry
- Biochemistry
Background:
- Dextransucrase enzymes synthesize dextran polysaccharides from sucrose.
- Previous research suggested D-glucose and sucrose act as initiator primers in dextran synthesis.
Purpose of the Study:
- To investigate the role of D-glucose and sucrose as initiator primers in dextran synthesis by Escherichia coli dextransucrase.
- To elucidate the mechanism of dextran polymerization and the role of conserved active site residues.
Main Methods:
- Utilized (14)C-labeled D-glucose and sucrose in enzymatic reactions.
- Performed chemical and enzymatic treatments of synthesized dextran.
- Analyzed dextran structure and incorporated radioactivity using pulse-chase experiments and acid hydrolysis.
Main Results:
- D-glucose and sucrose were found not to be initiator primers (<0.02% incorporation).
- Dextran chains are covalently attached to the dextransucrase active site during polymerization.
- (14)C-glucitol was detected in pulsed dextran, indicating D-glucose moieties are added to the reducing-ends of growing chains.
- Dextran molecular size is inversely proportional to enzyme concentration, suggesting a highly processive mechanism.
- Three conserved active site amino acids (Asp551, Glu589, Asp622) are implicated in the polymerization mechanism.
Conclusions:
- Escherichia coli dextransucrase synthesizes dextran by adding D-glucose moieties from sucrose to the reducing-ends of growing, covalently linked chains.
- The enzyme exhibits a highly processive mechanism involving a two-catalytic-site insertion model.
- Conserved active site residues play a crucial role in the catalytic mechanism of glucansucrases.
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