Volume 272, Number 35, Issue of August 29, 1997 pp. 21855-21864
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of the Gene Encoding the Escherichia coli Lipid A 4-Kinase
FACILE PHOSPHORYLATION OF ENDOTOXIN ANALOGS WITH RECOMBINANT LpxK

(Received for publication, April 28, 1997, and in revised form, June 20, 1997)

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

The genes for seven of nine enzymes needed for the biosynthesis of Kdo₂-lipid A (Re endotoxin) in Escherichia coli have been reported. We have now identified a novel gene encoding the lipid A 4-kinase (the sixth step of the pathway). The 4-kinase transfers the -phosphate of ATP to the 4-position of a tetraacyldisaccharide 1-phosphate intermediate (termed DS-1-P) to form tetraacyldisaccharide 1,4-bis-phosphate (lipid IV_A). The 4-phosphate is required for the action of distal enzymes, such as Kdo transferase and also renders lipid A substructures active as endotoxin antagonists or mimetics. Lysates of E. coli generated using individual  clones from the ordered Kohara library were assayed for overproduction of 4-kinase. Only one clone, [218]E1D1, which directed 2-2.5-fold overproduction, was identified. This construct contains 20 kilobase pairs of E. coli DNA from the vicinity of minute 21. Two genes related to the lipid A system map in this region: msbA, encoding a putative translocator, and kdsB, the structural gene for CMP-Kdo synthase. msbA forms an operon with a downstream, essential open reading frame of unknown function, designated orfE. orfE was cloned into a T7 expression system. Washed membranes from cells overexpressing orfE display ~2000-fold higher specific activity of 4-kinase than membranes from cells with vector alone. Membranes containing recombinant, overexpressed 4-kinase (but not membranes with wild-type kinase levels) efficiently phosphorylate three DS-1-P analogs: 3-aza-DS-1-P, base-treated DS-1-P, and base-treated 3-aza-DS-1-P. A synthetic hexaacylated DS-1-P analog, compound 505, can also be phosphorylated by membranes from the overproducer, yielding [4-³²P] lipid A (endotoxin). The overexpressed lipid A 4-kinase is very useful for making new 4-phosphorylated lipid A analogs with potential utility as endotoxin mimetics or antagonists. We suggest that orfE is the structural gene for the 4-kinase and that it be redesignated lpxK.

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