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J Biol Chem, Vol. 273, Issue 5, 2799-2807, January 30, 1998

Enzymatic Synthesis of Lipopolysaccharide in Escherichia coli
PURIFICATION AND PROPERTIES OF HEPTOSYLTRANSFERASE I

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

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, Kdo₂-lipid IV_A. Using a T7 promoter construct that overexpresses RfaC ~15,000-fold, the enzyme has been purified to near homogeneity. NH₂-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 mM for ADP-mannose and 4.5 µM for Kdo₂-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 Kdo₂-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 Kdo₂-lipid IV_A. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo₂-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 Kdo₂-[4-³²P]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.

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