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 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 gamma-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 IVA). 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 lambda 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 approximately 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.
Garrett
T A
TA
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Kadrmas
J L
JL
Raetz
C R
CR
D001426:Bacterial Proteins / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D005798:Genes, Bacterial; D008050:Lipid A / Q000031:analogs & derivatives* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D016366:Open Reading Frames; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D011994:Recombinant Proteins
D001426:Bacterial Proteins; D008050:Lipid A; D008070:Lipopolysaccharides; C085483:Re lipopolysaccharide; D011994:Recombinant Proteins; C088602:3-deoxy-2-octulosonic acid(2)-lipid IV(A); D017853:Phosphotransferases (Alcohol Group Acceptor); C076042:tetraacyldisaccharide 4'-kinase