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 lipopolysaccharide. The gene was subcloned from plasmid pWSB33 [Brabetz, W., Müller-Loennies, S., Holst, O. & Brade, H. (1997) Eur. J. Biochem. 247, 716-724] into a shuttle vector for the expression in the gram-positive host Corynebacterium glutamicum. The in vitro activity of the enzyme was investigated in comparison to that of heptosyltransferase I (WaaC) using as a source for the sugar nucleotide donor, ADP-LglyceroDmanno-heptose, a low molecular mass filtrate from a DeltawaaCF E. coli strain. Synthetic lipid A analogues varying in the acylation or phosphorylation pattern or both were tested as acceptors for the subsequent transfer of 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) and heptose by successive action of Kdo transferase (WaaA), heptosyltransferase I (WaaC) and heptosyltransferase II (WaaF). The reaction products were characterized after separation by TLC and blotting with monoclonal antibodies specific for the acceptor, the intermediates and the final products.
Gronow
S
S
Division of Medical and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany. sgronow@fz-borstel.de
Brabetz
W
W
Brade
H
H
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003352:Corynebacterium / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016695:Glycosyltransferases / Q000378:metabolism*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D008070:Lipopolysaccharides; D016695:Glycosyltransferases; C086735:heptosyltransferase; C080934:lipooligosaccharide 1,5-heptosyltransferase