A novel two‐gene requirement for the octanoyltransfer reaction of Bacillus subtilis lipoic acid biosynthesis

Abstract

SUMMARY The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM, and ywfL which we have renamed lplJ, lipM and lipL, respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to E. coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulfur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilis ΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coli ΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion paper (Christensen et al., 2011) provide evidence that LipM and LipL catalyze sequential reactions in a novel pathway for lipoic acid biosynthesis.

The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains

“…Complementation was tested on M9 minimal agar plates with glycerol as the sole carbon source to avoid bypass of succinate-and acetate-dependent growth by fermentative metabolism (33). Due to the E. coli QC146 ⌬lplA mutation, the strain is unable to grow with lipoic acid supplementation; however, growth proceeds robustly when a plasmid encoding lipoate ligase activity is present (5,10,13). Upon expression of the putative S. coelicolor lplA-expressing gene, the E. coli strain grew well, but only when the medium contained lipoic acid (Fig.…”

“…Although the function and protein structure of the E. coli LplA lipoate ligase are well established (4,5,7,11), the presence of this activity in other bacteria has been demonstrated only in B. subtilis, in which the ligase is called LplJ (10), and in Listeria monocytogenes, a bacterium related to B. subtilis that is auxotrophic for lipoic acid and that encodes two ligases, both of which function in lipoic acid scavenging (12). Like B. subtilis, Streptomyces coelicolor is a Gram-positive soil bacterium.…”

“…Two different bacterial lipoate synthesis pathways have been described: the E. coli LipB-LipA pathway and a more complex pathway in Bacillus subtilis that requires two additional proteins (9,10). Although the function and protein structure of the E. coli LplA lipoate ligase are well established (4,5,7,11), the presence of this activity in other bacteria has been demonstrated only in B. subtilis, in which the ligase is called LplJ (10), and in Listeria monocytogenes, a bacterium related to B. subtilis that is auxotrophic for lipoic acid and that encodes two ligases, both of which function in lipoic acid scavenging (12).…”

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

“…Biochemical assays showed that the LipM protein had no ligase activity and instead catalyzed octanoyl transfer from octanoyl-ACP (87). Another of the putative ligase genes (now called LplJ) was shown to encode a protein that functionally replaced LplA in E. coli and had only ligase activity (118). However, the protein (called LipL) encoded by the third putative lipoate ligase gene had no octanoyltransferase or ligase activity.…”

“…First, B. subtilis strains lacking the LplJ ligase should show a growth defect only in strains that also lack LipA. This was the case (118). Second, strains lacking the LipM octanoyltransferase or the LipL amidotransferase should require lipoic acid for growth in the absence of the products of the 2-oxoacid dehydrogenases.…”