Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase i.
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 approximately 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 microM for Kdo2-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 degrees 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.
Kadrmas
J L
JL
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Raetz
C R
CR
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000201:enzymology*; D006017:Glycolipids / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008050:Lipid A / Q000031:analogs & derivatives* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity
D006017:Glycolipids; D008050:Lipid A; D008070:Lipopolysaccharides; D009702:Nucleoside Diphosphate Sugars; D011994:Recombinant Proteins; C040142:lipid A precursors, bacterial; C088602:3-deoxy-2-octulosonic acid(2)-lipid IV(A); D017830:Octoxynol; D016695:Glycosyltransferases; C080934:lipooligosaccharide 1,5-heptosyltransferase