PMID:9268317: Difference between revisions

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<span style = 'font-size: large'> '''Garrett, TA, Kadrmas, JL and Raetz, CR'''  (1997) Identification of the gene encoding the Escherichia coli lipid A 4'-kinase. Facile phosphorylation of endotoxin analogs with recombinant LpxK.''J. Biol. Chem.'' '''272''':21855-64</span>
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'''Garrett, TA, Kadrmas, JL and Raetz, CR'''  (1997) Identification of the gene encoding the Escherichia coli lipid A 4'-kinase. Facile phosphorylation of endotoxin analogs with recombinant LpxK. ''J. Biol. Chem.'' '''272''':21855-64
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!align=left  |Abstract
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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.
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9268317 PubMed]


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|{{TableHeadingColor}} |'''Abstract''' || 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.
!align=left  |Keywords
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Bacterial Proteins/genetics; Chromosome Mapping; Cloning, Molecular; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression; Gene Library; Genes, Bacterial; Lipid A/analogs & derivatives; Lipid A/metabolism; Lipopolysaccharides/metabolism; Open Reading Frames; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor)/genetics; Recombinant Proteins
|{{TableHeadingColor}} nowrap |'''Links''' ||  [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9268317 PubMed]
 
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Latest revision as of 04:57, 15 September 2013

Citation

Garrett, TA, Kadrmas, JL and Raetz, CR (1997) Identification of the gene encoding the Escherichia coli lipid A 4'-kinase. Facile phosphorylation of endotoxin analogs with recombinant LpxK. J. Biol. Chem. 272:21855-64

Abstract

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.

Links

PubMed

Keywords

Bacterial Proteins/genetics; Chromosome Mapping; Cloning, Molecular; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression; Gene Library; Genes, Bacterial; Lipid A/analogs & derivatives; Lipid A/metabolism; Lipopolysaccharides/metabolism; Open Reading Frames; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor)/genetics; Recombinant Proteins

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