BIOSYNTHESIS OF LIPOPOLYSACCHARIDE IN ESCHERICHIA-COLI - CYTOPLASMIC ENZYMES THAT ATTACH 3-DEOXY-D-MANNO-OCTULOSONIC ACID TO LIPID-A

Authors:
  • KHK HOSAKA
    K HOSAKA
  • ARAD ROBERTSON
    AD ROBERTSON
  • CRCRH RAETZ
    CRH RAETZ
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  • 25 April 1989
    • journal article
    • research article
    • Vol. 264  (12) , 6956-6966
    • PMID:2651435
Abstract
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.degree.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-transfereases 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 lipolysaccharide substrates and analogs.

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