Biosynthesis of endotoxins. Purification and catalytic properties of 3-deoxy-D-manno-octulosonic acid transferase from Escherichia coli.
The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene of Escherichia coli and plays a key role in lipopolysaccharide biosynthesis. It transfers Kdo from CMP-Kdo to lipid A or its tetraacyldisaccharide-1,4'-bisphosphate precursor, lipid IVA. Using a strain that overproduces the transferase approximately 500-fold, we have purified the enzyme to near homogeneity. The subunit molecular mass is approximately 43 kDa. Activity is stimulated by Triton X-100, is maximal at pH 7, but does not require Mg2+. The apparent Km values for lipid IVA and CMP-Kdo are 52 and 88 microM, respectively. Vmax is 15-18 mumol/min/mg when both substrates are added near saturation at pH 8. The purified enzyme transfers 2 Kdo residues to lipid A precursors or analogs bearing four to six fatty acyl chains and a 4'-monosphosphate moiety. Activity is inhibited by polymixin B and Re endotoxin. At low Kdo concentrations small amounts of the intermediate, (Kdo)1-IVA, accumulate. When this substance is isolated and incubated with purified enzyme in the presence of CMP-Kdo, it is converted to (Kdo)2-IVA. Formation of (Kdo)1-IVA is also observed when purified enzyme is incubated with (Kdo)2-IVA and 5 mM CMP, demonstrating that Kdo transfer is reversible. In summary, Kdo transferase consists of a single bifunctional polypeptide that incorporates the 2 innermost Kdo residues common to all lipopolysaccharide molecules in E. coli.
Belunis
C J
CJ
Department of Biochemistry, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065.
Raetz
C R
CR
D001483:Base Sequence; D002240:Carbohydrate Sequence; D004731:Endotoxins / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005838:Genotype; D007700:Kinetics; D008050:Lipid A / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000096:biosynthesis; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D013379:Substrate Specificity; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004731:Endotoxins; D008050:Lipid A; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides; D014166:Transferases; C019917:3-deoxy-D-manno-octulosonate transferase