Biosynthesis of lipopolysaccharide in Escherichia coli. Cytoplasmic enzymes that attach 3-deoxy-D-manno-octulosonic acid to lipid A.
Previous studies in our laboratory led to the elucidation of the covalent structure of a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A (designated lipid IVA), that accumulates at 42 degrees C in temperature-sensitive mutants defective in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). Using [4'-32P]lipid IVA as the probe, we now demonstrate the existence of cytoplasmic KDO-transferases in Escherichia coli capable of attaching 2 KDO residues, derived from CMP-KDO, to lipid IVA. A partial purification has been developed to obtain a cytoplasmic subfraction that adds these 2 KDO residues with a 90% yield. The product is shown to have the stoichiometry of (KDO)2-IVA by fast atom bombardment mass spectrometry and NMR spectroscopy. The partially purified enzyme can utilize alternative lipid-disaccharide cosubstrates bearing five or six fatty acyl chains, but it has an absolute requirement for a monophosphate residue at position 4' of the lipid acceptor. When reincubated with a crude cytoplasmic fraction, a nucleoside triphosphate and Mg2+, (KDO)2-IVA is rapidly metabolized to more polar substances, the identity of which is unknown. The KDO-transferase(s) described in the present study should be very useful for the semisynthetic preparation of complex lipopolysaccharide substructures and analogs.
Brozek
K A
KA
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Hosaka
K
K
Robertson
A D
AD
Raetz
C R
CR
D003570:Cytidine Triphosphate / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D008050:Lipid A / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D009682:Magnetic Resonance Spectroscopy; D013379:Substrate Specificity; D013400:Sugar Acids / Q000378:metabolism*; D013997:Time Factors; D014166:Transferases / Q000378:metabolism*
D008050:Lipid A; D008070:Lipopolysaccharides; D013400:Sugar Acids; C002532:2-keto-3-deoxyoctonate; D003570:Cytidine Triphosphate; D014166:Transferases; C019917:3-deoxy-D-manno-octulosonate transferase