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J Biol Chem, Vol. 275, Issue 15, 11002-11009, April 14, 2000

Antibacterial Agents That Target Lipid A Biosynthesis in Gram-negative Bacteria
INHIBITION OF DIVERSE UDP-3-O-(R-3-HYDROXYMYRISTOYL)-N-ACETYLGLUCOSAMINE DEACETYLASES BY SUBSTRATE ANALOGS CONTAINING ZINC BINDING MOTIFS^*

Jane E. Jackman^§, Carol A. Fierke^¶, L. Nathan Tumey, Michael Pirrung, Taketo Uchiyama^**, S. Hasan Tahir^**, Ole Hindsgaul^**, and Christian R. H. Raetz

From the Departments of  Biochemistry and  Chemistry, Duke University Medical Center, Durham, North Carolina 27710 and the ^** Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A, a unique amphiphilic molecule found in the outer membranes of virtually all Gram-negative bacteria. Since lipid A biosynthesis is required for bacterial growth, inhibitors of LpxC have potential utility as antibiotics. The enzymes of lipid A biosynthesis, including LpxC, are encoded by single copy genes in all sequenced Gram-negative genomes. We have now cloned, overexpressed, and purified LpxC from the hyperthermophile Aquifex aeolicus. This heat-stable LpxC variant (the most divergent of all known LpxCs) displays 32% identity and 51% similarity over 277 amino acid residues out of the 305 in Escherichia coli LpxC. Although A. aeolicus LpxC deacetylates the substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine at a rate comparable with E. coli LpxC, a phenyloxazoline-based hydroxamate that inhibits E. coli LpxC with K_i of ~50 nM (Onishi, H. R., Pelak, B. A., Gerckens, L. S., Silver, L. L., Kahan, F. M., Chen, M. H., Patchett, A. A., Galloway, S. M., Hyland, S. A., Anderson, M. S., and Raetz, C. R. H. (1996) Science 274, 980-982) does not inhibit A. aeolicus LpxC. To determine whether or not broad-spectrum deacetylase inhibitors can be found, we have designed a new class of hydroxamate-containing inhibitors of LpxC, starting with the structure of the physiological substrate. Several of these compounds inhibit both E. coli and A. aeolicus LpxC at similar concentrations. We have also identified a phosphinate-containing substrate analog that inhibits both E. coli and A. aeolicus LpxC, suggesting that the LpxC reaction proceeds by a mechanism similar to that described for other zinc metalloamidases, like carboxypeptidase A and thermolysin. The differences between the phenyloxazoline and the substrate-based LpxC inhibitors might be exploited for developing novel antibiotics targeted either against some or all Gram-negative strains. We suggest that LpxC inhibitors with antibacterial activity be termed "deacetylins."

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^* This work was supported by National Institutes of Health Grants GM51310 (to C. R. H. R.), GM40602 (to C. A. F.), and AI 42141 (to M. P.). Glycobiotics Inc. (Athens, GA) funded the synthesis of the substrate analog inhibitors of LpxC in the laboratory of O. H.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported in part by a National Science Foundation predoctoral fellowship.

^¶ Present address: Dept. of Chemistry, University of Michigan, Ann Arbor, MI 48109.

To whom correspondence should be addressed. E-mail: Raetz@biochem.duke.edu.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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