Utilization of an undecaprenol kinase for investigations of oligosaccharide transfer

BIOL 106

Meredith D Hartley, mhartley@mit.edu, Department of Chemistry, Massachusetts Institue of Technology, 77 Massachusetts Ave., 18-543, Cambridge, MA 02139, Angelyn K. Larkin, alarkin@mit.edu, Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., 18-563, Cambridge, MA 02139, and Barbara Imperiali, imper@mit.edu, Department of Chemistry and Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139.
To elucidate the initial step in the N-linked glycosylation pathway in Campylobacter jejuni, two undecaprenol kinase candidates were identified. Streptococcus mutans diacylglycerol kinase (dgk) has been previously shown to have undecaprenol kinase activity and C. jejuni dgk was identified based on sequence homology. The two enzymes were cloned from their respective genomes and expressed heterologously in Escherichia coli. Radioactivity-based assays in which phosphorylation of undecaprenol was coupled to PglC and PglA, glycosyltransferases from C. jejuni, revealed that S. mutans dgk was very active, while the C. jejuni enzyme had relatively low activity. Using the kinase for the initial phosphorylation circumvents a challenging chemical phosphorylation on limited quantities of polyprenol and results in higher yields of the desired radiolabeled undecaprenyl pyrophosphate-linked disaccharide. This intermediate in the pathway is the minimal substrate for the C. jejuni oligosaccharyl transferase, PglB, and has been utilized in our ongoing studies of the enzymatic mechanism.

 

Frontiers in Chemical Biology
5:00 PM-7:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Biological Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007