Identification of a novel lipoic acid biosynthesis pathway reveals the complex evolution of lipoate assembly in prokaryotes.

Lipoic acid is an essential biomolecule found in all domains of life and is involved in central carbon metabolism and dissimilatory sulfur oxidation. The machineries for lipoate assembly in mitochondria and chloroplasts of higher eukaryotes, as well as in the apicoplasts of some protozoa, are all of prokaryotic origin. Here, we provide experimental evidence for a novel lipoate assembly pathway in bacteria based on a sLpl(AB) lipoate:protein ligase, which attaches octanoate or lipoate to apo-proteins, and 2 radical SAM proteins, LipS1 and LipS2, which work together as lipoyl synthase and insert 2 sulfur atoms. Extensive homology searches combined with genomic context analyses allowed us to precisely distinguish between the new and established pathways and map them on the tree of life. This not only revealed a much wider distribution of lipoate biogenesis systems than expected, in particular, the novel sLpl(AB)-LipS1/S2 pathway, and indicated a highly modular nature of the enzymes involved, with unforeseen combinations, but also provided a new framework for the evolution of lipoate assembly. Our results show that dedicated machineries for both de novo lipoate biogenesis and scavenging from the environment were implemented early in evolution and that their distribution in the 2 prokaryotic domains was shaped by a complex network of horizontal gene transfers, acquisition of additional genes, fusions, and losses. Our large-scale phylogenetic analyses identify the bipartite archaeal LplAB ligase as the ancestor of the bacterial sLpl(AB) proteins, which were obtained by horizontal gene transfer. LipS1/S2 have a more complex evolutionary history with multiple of such events but probably also originated in the domain archaea.

Fe/S proteins in microbial sulfur oxidation.

Iron-sulfur clusters serve as indispensable cofactors within proteins across all three domains of life. Fe/S clusters emerged early during the evolution of life on our planet and the biogeochemical cycle of sulfur is one of the most ancient and important element cycles. It is therefore no surprise that Fe/S proteins have crucial roles in the multiple steps of microbial sulfur metabolism. During dissimilatory sulfur oxidation in prokaryotes, Fe/S proteins not only serve as electron carriers in several steps, but also perform catalytic roles, including unprecedented reactions. Two cytoplasmic enzyme systems that oxidize sulfane sulfur to sulfite are of particular interest in this context: The rDsr pathway employs the reverse acting dissimilatory sulfite reductase rDsrAB as its key enzyme, while the sHdr pathway utilizes polypeptides resembling the HdrA, HdrB and HdrC subunits of heterodisulfide reductase from methanogenic archaea. Both pathways involve components predicted to bind unusual noncubane Fe/S clusters acting as catalysts for the formation of disulfide or sulfite. Mapping of Fe/S cluster machineries on the sulfur-oxidizing prokaryote tree reveals that ISC, SUF, MIS and SMS are all sufficient to meet the Fe/S cluster maturation requirements for operation of the sHdr or rDsr pathways. The sHdr pathway is dependent on lipoate-binding proteins that are assembled by a novel pathway, involving two Radical SAM proteins, namely LipS1 and LipS2. These proteins coordinate sulfur-donating auxiliary Fe/S clusters in atypical patterns by three cysteines and one histidine and act as lipoyl synthases by jointly inserting two sulfur atoms to an octanoyl residue. This article is part of a Special Issue entitled: Biogenesis and Function of Fe/S proteins.