Identification of a UDP-Gal: GlcNAc-R galactosyltransferase activity in Escherichia coli VW187

• Published in: Bioorganic & medicinal chemistry letters 2005-02, Vol.15 (4), p.1205-1211
• Main Authors: Montoya-Peleaz, Pedro J., Riley, John G., Szarek, Walter A., Valvano, Miguel A., Schutzbach, John S., Brockhausen, Inka
• Format: Article
• Language: English
• Subjects: Bacteriology; Biological and medical sciences; Enzyme assay; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Galactosyltransferase; Galactosyltransferases - metabolism; Glucosyltransferases; Humans; Metabolism. Enzymes; Microbiology; O-Antigen synthesis; Structure-Activity Relationship; Substrate Specificity; Uridine Diphosphate Galactose - metabolism
• ISSN: 0960-894X; 1464-3405
• DOI: 10.1016/j.bmcl.2004.11.077

The paper reports the synthesis of a GlcNAc α-pyrophosphate, covalently bound to a phenoxyundecyl moiety, and its use as an acceptor substrate in a novel assay for a galactosyltransferase from Escherichia coli. A novel acceptor substrate for galactosyltransferase was synthesized containing GlcNAcα-pyrophosphate, covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc α-O-PO 3-PO 3-(CH 2) 11-O-Phenyl). The new substrate was used to develop an assay for a galactosyltransferase activity from Escherichia coli strain VW187 that is involved in lipopolysaccharide synthesis and has not been studied by others. We showed that Gal was transferred from UDP-Gal to the novel acceptor substrate. This was a significant improvement over our previous preliminary assays of the enzyme using endogenous substrate, and showed that these synthetic substrates are useful for assaying enzymes that utilize lipid-bound substrates in O-chain synthesis in Gram-negative bacteria.