PMID:1577828
| Citation |
Belunis, CJ and Raetz, CR (1992) Biosynthesis of endotoxins. Purification and catalytic properties of 3-deoxy-D-manno-octulosonic acid transferase from Escherichia coli. J. Biol. Chem. 267:9988-97 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Base Sequence; Carbohydrate Sequence; Endotoxins/biosynthesis; Escherichia coli/enzymology; Escherichia coli/genetics; Genes, Bacterial; Genotype; Kinetics; Lipid A/biosynthesis; Lipopolysaccharides/biosynthesis; Models, Biological; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Polymerase Chain Reaction/methods; Restriction Mapping; Substrate Specificity; Transferases/genetics; Transferases/isolation & purification; Transferases/metabolism |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.
