A Leptospira interrogans enzyme with similarity to yeast Ste14p that methylates the 1-phosphate group of lipid A

Middleton Boon Hinckley, C. Michael Reynolds, Anthony A. Ribeiro, Sara C. McGrath, Robert J. Cotter, Fanny N. Lauw, Douglas T. Golenbock, Christian R.H. Raetz

Research output: Contribution to journalArticle

Abstract

Distinct from other spirochetes, cells of Leptospira interrogans contain orthologues of all the Escherichia coli lpx genes required for lipid A biosynthesis, but they synthesize a modified form of lipopolysaccharide that supposedly activates toll-like receptor 2 (TLR2) instead of TLR4. The recent determination of the L. interrogans lipid A structure revealed an unprecedented O-methylation of its 1-phosphate group (Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25420-25429). The enzymatic activity responsible for selective 1-phosphate methylation has not been previously explored. A membrane enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the 1-phosphate moiety of E. coli Kdo2-[4′-32P]lipid A has now been discovered. The gene encoding this enzyme was identified based on the hypothesis that methylation of a phosphate group is chemically analogous to methylation of a carboxylate moiety at a membrane-water interface. Database searching revealed a candidate gene (renamed lmtA) in L. interrogans showing distant homology to the yeast isoprenylcysteine carboxyl methyltransferase, encoded by sterile-14, which methylates the a-type mating factor. Orthologues of lmtA were not present in E. coli, the lipid A of which normally lacks the 1-phosphomethyl group, or in other spirochetes, which do not synthesize lipid A. Expression of the lmtA gene behind the lac promoter on a low copy plasmid resulted in the appearance of SAM-dependent methyltransferase activity in E. coli inner membranes and methylation of about 30% of the endogenous E. coli lipid A. Inactivation of the ABC transporter MsbA did not inhibit methylation of newly synthesized lipid A. Methylated E. coli lipid A was analyzed by mass spectrometry and NMR spectroscopy to confirm the location of the phosphomethyl group at the 1-position. In human cells, engineered to express the individual TLR subtypes, 1-phosphomethyl-lipid A purified from lmtA-expressing E. coli potently activated TLR4 but not TLR2.

Original languageEnglish (US)
Pages (from-to)30214-30224
Number of pages11
JournalJournal of Biological Chemistry
Volume280
Issue number34
DOIs
StatePublished - Aug 26 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Hinckley, M. B., Reynolds, C. M., Ribeiro, A. A., McGrath, S. C., Cotter, R. J., Lauw, F. N., Golenbock, D. T., & Raetz, C. R. H. (2005). A Leptospira interrogans enzyme with similarity to yeast Ste14p that methylates the 1-phosphate group of lipid A. Journal of Biological Chemistry, 280(34), 30214-30224. https://doi.org/10.1074/jbc.M506103200