Abstract

Lipoate is an essential cofactor for key enzymes of oxidative and one-carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase (DH) complexes and the GcvH subunit of the glycine cleavage system. Bacillus subtilis possess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, amidotransfer of the octanoate to E2s, and insertion of sulfur atoms. Lipoate scavenging is mediated by a lipoate ligase (LplJ), that catalizes a classical two-step ATP-dependent reaction. Although these pathways were thought to be redundant, a {Delta}lipL mutant, unable to transfer the octanoyl group from GcvH to the E2s during lipoate synthesis, showed growth defects in minimal media even when supplemented with this cofactor, despite the presence of a functional LplJ. In this study we demonstrated that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate DH during lipoate scavenging. LipL must be functional and it is not forming a complex with LplJ, which suggests that these enzymes might be acting sequentially. We also show that the E2 subunit of oxoglutarate DH is a good donor for LipL amidotransfer reaction. The essential role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. LplJ does not recognize E2 subunits without a negatively charged residue in key positions of the target protein, and thus LipL is required to transfer the lipoate to them. This model of lipoate scavenging seems widespread among Gram-positive bacteria.