Scavenging of cytosolic octanoic acid by mutant LplA lipoate ligases allows growth of Escherichia coli strains lacking the LipB octanoyltransferase of lipoic acid synthesis

Abstract

The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in Escherichia coli, transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid dehydrogenases of aerobic metabolism. Strains containing null mutations in lipB are auxotrophic for either lipoic acid or octanoic acid. We report the isolation of two spontaneously arising mutant strains that allow growth of lipB strains on glucose minimal medium; we determined that suppression was caused by single missense mutations within the coding sequence of the gene (lplA) that encodes lipoate-protein ligase. The LplA proteins encoded by the mutant genes have reduced K(m) values for free octanoic acid and thus are able to scavenge cytosolic octanoic acid for octanoylation of lipoyl domains.

FIG. 1.

Lipoic acid metabolism in E. coli. (A) LplA lipoate ligase reaction, in which lipoate reacts with ATP to form the activated intermediate, lipoyl-adenylate (lipoyl-AMP), which remains firmly bound within the active site. The lipoyl-adenylate mixed anhydride bond is then attacked by the ɛ-amino group of the target lysine residue of the acceptor lipoyl domain to form lipoylated protein. LplA also utilizes octanoic acid. (B) Lipoic acid synthesis in E. coli. LipB transfers an octanoyl moiety from the fatty acid biosynthetic intermediate, octanoyl-ACP, to the lipoyl domain of a lipoate-accepting protein (in this case the E2 subunit of a 2-oxoacid dehydrogenase). The octanoylated domain is the substrate of LipA, an S-adenosylmethionine radical enzyme that replaces one hydrogen atom on each of octanoate carbons 6 and 8 with sulfur atoms. For a review, see reference .

FIG. 2.

PDH (solid bars) and OGDH (open bars) activities of various isogenic strains grown on glucose minimal medium supplemented with succinate and acetate. The values reported are the averages of three measurements. Strain JK1, wild type; strain ZX221, lipB; strain FH145, lipB lplA10; and strain FH146, lipB lplA11.

FIG. 3.

Western blot analysis of protein lipoylation. All strains were isogenic and produced a hybrid lipoyl domain encoded by plasmid pGS331 (1). Equal amounts of total extract protein were loaded in each lane. In panel A, the cells were from cultures grown without octanoic acid supplementation, whereas in panel B, the cultures were grown with octanoic acid supplementation. Lane 1, lipoyl domain standard; lane 2, strain JK1 (wild type); lane 3, strain TM135 (lplA); lane 4, strain ZX221 (lipB); lane 5, strain FH145 (lipB lplA10); lane 6, strain FH146 (lipB lplA11).

FIG. 4.

Gel shift assay with V19L LplA and octanoyl-ACP as a substrate. Two preparations of octanoyl-ACP (undialyzed and dialyzed) were tested. Lanes 3, 5, 7, and 9 were controls for the integrity of the octanoyl-ACP thioester bond in both preparations because LipB cannot utilize free octanoic acid. In these four lanes, the modified domain migrates more rapidly than the unmodified domain. In the LipB reactions, a holo-ACP band is seen at the top of the gel. Lanes 2 and 4 show V19L LplA-catalyzed modification of the domain in the presence of an undialyzed octanoyl-ACP preparation. However, there was no accumulation of holo-ACP, indicating that octanoyl-ACP was not the source of octanoate. In lane 6, an extensively dialyzed octanoyl-ACP preparation was the substrate, and no modification of the domain was seen. In lane 8, ATP was added, which resulted in some modification of the domain, which may be due to hydrolysis of octanoyl-ACP to give free octanoate plus Mg²⁺ introduced with the octanoyl-ACP (ACP is known to avidly bind Mg²⁺, which was present at a high concentration during octanoyl-ACP synthesis). The S221P LplA protein gave essentially identical results.

FIG. 5.

Gas chromatogram of the butyl esters of free fatty acid from cell extracts. The internal-standard butyl-heptanoate peak represents 50 ng. The mass spectra of the butyl esters of heptanoate, octanoate, and decanoate were identical to those of authentic standards and the database entries.