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J. Biol. Chem., Vol. 280, Issue 34, 30214-30224, August 26, 2005

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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||, and Christian R. H. Raetz

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, Duke NMR Spectroscopy Center, Duke University Medical Center, Durham, North Carolina 27710, ^¶Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the ^||Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605

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.

Received for publication, June 3, 2005 , and in revised form, June 27, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s)DQ097086.

^* This research was funded by National Institutes of Health (NIH) Grants 1-F32-GM073251-01 (to M. B. H.), GM-51310 and GM-51796 (to C. R. H. R.), GM-54882 (to R. J. C.), and GM-54060 and AI057784 (to D. T. G.). The Duke NMR Center is partially supported by NCI, NIH, Grant P30-CA-14236. Duke NMR Center instrumentation was funded by the National Science Foundation, the NIH, the North Carolina Biotechnology Center, and Duke University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Table I.

^** Supported by a grant from the Netherlands Organization for Scientific Research.

To whom correspondence should be addressed. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz{at}biochem.duke.edu.

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