Enzymatic Synthesis of Lipopolysaccharide in Escherichia coli

• Published in: The Journal of biological chemistry 1998-01, Vol.273 (5), p.2799-2807
• Main Authors: Kadrmas, Julie L., Raetz, Christian R.H.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.5.2799

Heptosyltransferase I, encoded by the rfaC ( waaC ) gene of Escherichia coli , is thought to add l - glycero - d - manno -heptose to the inner 3-deoxy- d - manno -octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP- l - glycero - d - manno -heptose, has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo 2 -lipid IV A . Using a T7 promoter construct that overexpresses RfaC ∼15,000-fold, the enzyme has been purified to near homogeneity. NH 2 -terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa. Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparent K m (determined at near saturating concentrations of the second substrate) is 1.5 m m for ADP-mannose and 4.5 μ m for Kdo 2 -lipid IV A . Chemical hydrolysis of the RfaC reaction product at 100 °C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo 2 -lipid IV A as the site of mannosylation. The analog, Kdo-lipid IV A , functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo 2 -lipid IV A . The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo 2 -lipid IV A catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo 2 -[4′- 32 P]lipid IV A to assay for the physiological donor (presumably ADP- l - glycero - d - manno -heptose) in a crude, low molecular weight fraction isolated from wild type cells.