Comparative functional characterization in vitro of heptosyltransferase I (WaaC) and II (WaaF) from Escherichia coli

Heptosyltransferase II, encoded by the waaF gene of Escherichia coli, is a glycosyltransferase involved in the synthesis of the inner core region of l ipopolysaccharide. The gene was subcloned from plasmid pWSB33 [Brabetz, W., Muller-Loennies, S., Holst, O. & Brade, H. (1997) Eur. J. Biochem. 247, 71 6-724] into a shuttle vector for the expression in the gram-positive host C orynebacterium glutamicum. The in vitro activity of the enzyme was investig ated in comparison to that of heptosyltransferase I (WaaC) using as a sourc e for the sugar nucleotide donor, ADP-L-glycero-D-manno-heptose, a low mole cular mass filtrate from a Delta waaCF E. coli strain. Synthetic lipid A an alogues varying in the acylation or phosphorylation pattern or both were te sted as acceptors for the subsequent transfer of 3-deoxy-D-manno-oct-2-ulos onic acid (Kdo) and heptose by successive action of Kdo transferase (WaaA), heptosyltransferase I (WaaC) and heptosyltransferase II (WaaF). The reacti on products were characterized after separation by TLC and blotting with mo noclonal antibodies specific for the acceptor, the intermediates and the fi nal products.