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J Biol Chem, Vol. 273, Issue 5, 2799-2807, January 30, 1998
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 IVA. 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
Km (determined at near saturating concentrations of
the second substrate) is 1.5 mM for ADP-mannose and 4.5 µM for Kdo2-lipid IVA. 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
IVA as the site of mannosylation. The analog, Kdo-lipid
IVA, functions as an acceptor, but is mannosylated at less
than 1% the rate of Kdo2-lipid IVA. The
purified enzyme displays no activity with ADP-glucose, GDP-mannose,
UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid
IVA 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
IVA 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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