Bacterial lipoate synthesis revisited: novel enzymes, unusual substrates and new evolutionary perspectives

• Applicant: Privatdozentin Dr. Christiane Dahl
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525834735

Lipoic acid is present in organisms throughout all domains of life and involved in key reactions of central carbon metabolism and dissimilatory sulfur oxidation. In this eight-carbon saturated fatty acid, sulfur atoms replace the hydrogen atoms of carbons 6 and 8 of the acyl chain. Two posttranslational machineries for synthesis of lipoyl moieties on their target proteins are well characterized: Salvage of free precursors or de novo synthesis from intermediates of fatty acid biosynthesis. Recently, a novel pathway for the assembly of this important biomolecule has been discovered, which is widely distributed in both prokaryotic domains. Previously, lipoate:protein ligases had been thought to exclusively operate in lipoate salvage. However, our work on bacterial sulfur oxidizers shows that they contain ligases involved in de novo lipoylation of proteins, starting from octanoate as a free precursor. Lipoate assembly is then completed by the incorporation of sulfur via two radical SAM domain-containing proteins, LipS1 and LipS2. For the archaeon Thermococcus kodakarensis the pathway has recently been experimentally demonstrated, while corresponding work on representatives of the domain Bacteria is still pending. Our goal is to characterize the novel bacterial lipoate assembly pathway in detail by addressing the following major points: 1. Identification of all enzymes involved in the pathway. 2. Properties as well as interactions of the novel bacterial lipoate assembly proteins especially with respect to differences from their archaeal counterparts. 3. Substrate ranges and mechanisms of differentiation between substrates for lipoylation pathways running in parallel in the same organism. 4. Prevalence and general importance of the new lipoylation pathway with a focus on the domain Bacteria. 5. Origin and evolution of lipoate assembly machineries. Several different experimental approaches will be applied: Genetic studies will include gene inactivation and complementation in the Alphaproteobacterium Hyphomicrobium denitrificans. The native LbpA2 protein will be purified and analysed from H. denitrificans reference and mutant strains allowing us to assign individual assembly steps to individual enzymes. Detailed biochemical experiments on the pure recombinant proteins will be complemented by structural analyses and thus provide further insight into the function of the studied system. An in vitro system for lipoate assembly combining the new enzymes will be established using bacterial apo-LbpA proteins as the native substrate. Exhaustive large-scale database analyses will be performed using our new bioinformatic tool, HMS-S-S. We intend to map the novel lipoate synthesis pathway on the complete tree of life thus revealing its distribution. Finally, phylogenetic analyses will be performed in order to gather information on the evolution of the new synthesis pathway.

• DFG Programme: Research Grants