Function of the Escherichia coli msbB gene, a multicopy suppressor of htrB knockouts, in the acylation of lipid A. Acylation by MsbB follows laurate incorporation by HtrB.
Overexpression of the Escherichia coli msbB gene on high copy plasmids suppresses the temperature-sensitive growth associated with mutations in the htrB gene. htrB encodes the lauroyl transferase of lipid A biosynthesis that acylates the intermediate (Kdo)2-lipid IVA (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). Since msbB displays 27.5% identity and 42.2% similarity to htrB, we explored the possibility that msbB encodes a related acyltransferase. In contrast to htrB, extracts of strains with insertion mutations in msbB are not defective in transferring laurate from lauroyl acyl carrier protein to (Kdo)2-lipid IVA. However, extracts of msbB mutants do not efficiently acylate the product formed by HtrB, designated (Kdo)2-(lauroyl)-lipid IVA. Extracts of strains harboring msbB+ bearing plasmids acylate (Kdo)2-(lauroyl)-lipid IVA very rapidly compared with wild type. We solubilized and partially purified MsbB from an overproducing strain, lacking HtrB. MsbB transfers myristate or laurate, activated on ACP, to (Kdo)2-(lauroyl)-lipid IVA. Decanoyl, palmitoyl, palmitoleoyl, and (R)-3-hydroxymyristoyl-ACP are poor acyl donors. MsbB acylates (Kdo)2-(lauroyl)-lipid IVA about 100 times faster than (Kdo)2-lipid IVA. The slow, but measurable, rate whereby MsbB acts on (Kdo)2-lipid IVA may explain why overexpression of MsbB suppresses the temperature-sensitive phenotype of htrB mutations. Presumably, the acyloxyacyl group generated by excess MsbB substitutes for the one normally formed by HtrB.
Clementz
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Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Zhou
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Raetz
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CR
D000215:Acylation; D000217:Acyltransferases; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002474:Cell-Free System; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D016153:Genes, Suppressor; D005838:Genotype; D007850:Lauric Acids / Q000378:metabolism*; D008050:Lipid A / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007850:Lauric Acids; D008050:Lipid A; C030358:lauric acid; D000217:Acyltransferases; C097895:MsbB protein, E coli