IDENTIFICATION OF THE GENE ENCODING THE ESCHERICHIA-COLI LIPID A 4'-KINASE - FACILE PHOSPHORYLATION OF ENDOTOXIN ANALOGS WITH RECOMBINANT LPXK

The genes for seven of nine enzymes needed for the biosynthesis of Kdo (2)-lipid A (Re endotoxin) in Escherichia coli have been reported, We have now identified a novel gene encoding the lipid A 4'-kinase (the s ixth step of the pathway). The 4'-kinase transfers the (gamma) over do t-phosphate of ATP to the 4'-position of a tetraacyldisaccharide 1-pho sphate intermediate (termed DS-1-P) to form tetraacyldisaccharide 1,4' -bis-phosphate (lipid IVA). The 4'-phosphate is required for the actio n 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 A clones from the ordered Kohara li brary were assayed for over-production of 4'-kinase. Only one clone, [ 218]E1D1, which directed 2-2.5-fold overproduction, was identified. Th is construct contains 20 kilobase pairs of E. coli DNA from the vicini ty of minute 21. Two genes related to the lipid A system map in this r egion: msbA, encoding a putative translocator, and kdsB, the structura l gene for CMP-Kdo synthase, msbA forms an operon with a downstream, e ssential open reading frame of unknown function, designated orfE. orfE was cloned into a T7 expression system. Washed membranes from cells o verexpressing orfE display similar to 2000-fold higher specific activi ty of 4'-kinase than membranes from cells with vector alone. Membranes containing recombinant, overexpressed 4'-kinase (but not membranes wi th wild-type kinase levels) efficiently phosphorylate three DS-1-P ana logs: 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 ph osphorylated by membranes from the overproducer, yielding [4'-P-32] li pid A (endotoxin). The overexpressed lipid A 4'-kinase is very useful for making new 4'-phosphorylated lipid A analogs with potential utilit y as endotoxin mimetics or antagonists, We suggest that orfE is the st ructural gene for the 4'-kinase and that it be redesignated IpxK.