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Functional identification of bacterial glucosyltransferase WbdN
Yin Gao1, Anna Vinnikova, Inka Brockhausen
1Department of Medicine, Division of Rheumatology, Queen's University, Kingston, Ontario, Canada.
Methods in Molecular Biology (Clifton, N.J.)
|June 15, 2013
Summary
Researchers identified the function of a key enzyme, WbdN, involved in building bacterial outer membranes. This work utilizes novel NMR and fluorescent assays for enzyme characterization without radioactive materials.
Area of Science:
- Microbiology
- Biochemistry
- Glycobiology
Background:
- Gram-negative bacteria possess an outer membrane crucial for their structure and survival.
- Lipopolysaccharides (LPS), particularly their O-antigenic polysaccharides, stabilize this outer membrane.
- The biosynthesis of O-antigen repeating units involves glycosyltransferases acting on lipid-linked intermediates.
Purpose of the Study:
- To elucidate the enzymatic function of the glucosyltransferase WbdN from Escherichia coli O157.
- To develop and apply novel biochemical methods for characterizing glycosyltransferase activity.
- To enable the study of enzymes involved in LPS biosynthesis.
Main Methods:
- Utilized a recombinant glucosyltransferase (WbdN) from Escherichia coli O157.
- Employed Nuclear Magnetic Resonance (NMR) spectroscopy to analyze enzyme products.
- Developed and used a synthetic acceptor substrate analog for enzyme assays.
- Applied fluorescent acceptor substrate analogs for sensitive enzyme activity characterization.
Main Results:
- Successfully determined the enzymatic function of WbdN using NMR analysis of its product with a synthetic analog.
- Demonstrated the utility of fluorescent acceptor substrate analogs for sensitive enzyme assays.
- Showcased a method for characterizing enzyme activity without relying on radioactive nucleotide sugar donors.
Conclusions:
- The function of the Escherichia coli O157 glucosyltransferase WbdN has been identified.
- Novel NMR and fluorescence-based assays provide sensitive and efficient tools for studying glycosyltransferases.
- These methods facilitate the characterization of enzymes involved in lipopolysaccharide biosynthesis.
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