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An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A.

Publication ,  Journal Article
Gibbons, HS; Reynolds, CM; Guan, Z; Raetz, CRH
Published in: Biochemistry
March 4, 2008

The lipid A residues of certain Gram-negative bacteria, including most strains of Salmonella and Pseudomonas, are esterified with one or two secondary S-2-hydroxyacyl chains. The S-2 hydroxylation process is O 2-dependent in vivo, but the relevant enzymatic pathways have not been fully characterized because in vitro assays have not been developed. We previously reported that expression of the Salmonella lpxO gene confers upon Escherichia coli K-12 the ability to synthesize 2-hydroxymyristate modified lipid A ( J. Biol. Chem. (2000) 275, 32940-32949). We now demonstrate that inactivation of lpxO, which encodes a putative Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase, abolishes S-2-hydroxymyristate formation in S. typhimurium. Membranes of E. coli strains expressing lpxO are able to hydroxylate Kdo 2-[4'- (32)P]-lipid A in vitro in the presence of Fe (2+), O 2, alpha-ketoglutarate, ascorbate, and Triton X-100. The Fe (2+) chelator 2,2'-bipyridyl inhibits the reaction. The product generated in vitro is a monohydroxylated Kdo 2-lipid A derivative. The [4'- (32)P]-lipid A released by mild acid hydrolysis from the in vitro product migrates with authentic S-2-hydroxlyated lipid A isolated from (32)P-labeled S. typhimurium cells. Electrospray ionization mass spectrometry and gas chromatography/mass spectrometry of the in vitro product are consistent with the 2-hydroxylation of the 3'-secondary myristoyl chain of Kdo 2-lipid A. LpxO contains two predicted trans-membrane helices (one at each end of the protein), and its active site likely faces the cytoplasm. LpxO is an unusual example of an integral membrane protein that is a member of the Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase family.

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Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

March 4, 2008

Volume

47

Issue

9

Start / End Page

2814 / 2825

Location

United States

Related Subject Headings

  • Spectrometry, Mass, Electrospray Ionization
  • Salmonella typhimurium
  • Phospholipids
  • Mutation
  • Molecular Structure
  • Lipid A
  • Gas Chromatography-Mass Spectrometry
  • Escherichia coli
  • Dioxygenases
  • Biochemistry & Molecular Biology
 

Citation

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Gibbons, H. S., Reynolds, C. M., Guan, Z., & Raetz, C. R. H. (2008). An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A. Biochemistry, 47(9), 2814–2825. https://doi.org/10.1021/bi702457c
Gibbons, Henry S., C Michael Reynolds, Ziqiang Guan, and Christian R. H. Raetz. “An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A.Biochemistry 47, no. 9 (March 4, 2008): 2814–25. https://doi.org/10.1021/bi702457c.
Gibbons HS, Reynolds CM, Guan Z, Raetz CRH. An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A. Biochemistry. 2008 Mar 4;47(9):2814–25.
Gibbons, Henry S., et al. “An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A.Biochemistry, vol. 47, no. 9, Mar. 2008, pp. 2814–25. Pubmed, doi:10.1021/bi702457c.
Gibbons HS, Reynolds CM, Guan Z, Raetz CRH. An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A. Biochemistry. 2008 Mar 4;47(9):2814–2825.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

March 4, 2008

Volume

47

Issue

9

Start / End Page

2814 / 2825

Location

United States

Related Subject Headings

  • Spectrometry, Mass, Electrospray Ionization
  • Salmonella typhimurium
  • Phospholipids
  • Mutation
  • Molecular Structure
  • Lipid A
  • Gas Chromatography-Mass Spectrometry
  • Escherichia coli
  • Dioxygenases
  • Biochemistry & Molecular Biology