Novel lipoate-binding proteins and their role in sulfur oxidation

• Applicant: Privatdozentin Dr. Christiane Dahl
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433613342

Lipoic acid (1,2-dithiolane-3-pentanoic acid) is found in all domains of life and is involved in key reactions of central carbon metabolism. So far, only five lipoate-dependent multi¬enzyme complexes have been characterized: three α-ketoacid dehydrogenases (e.g. pyruvate dehydrogenase), acetoin dehydrogenase and the glycine cleavage complex. We found an additional function for this highly conserved organosulfur cofactor and demonstrated that novel lipoate-binding proteins (LbpA) act as indispensable components of a new pathway of dissimilatory sulfur oxidation. A heterodisulfide reductase-like complex (sHdr) is a central player in this pathway. It occurs in a large groups of organism including biotechnologically and environmentally relevant bacteria such as the volatile organic sulfur compound degrader Hyphomicrobium denitrificans and many chemolithoautotrophic bacteria and archaea. In the first phase of our project, we characterized a cascade of sulfur transferases that deliver sulfur to the sulfur-oxidizing sHdr-LbpA pathway. We discovered that LbpA proteins are matured by a novel assembly pathway. We collected initial evidence that LbpA proteins function as sulfur-binding entities that present substrate to the catalytic site(s) of the sulfur-oxidizing sHdr complex. However, a completely conclusive picture of LbpA function cannot yet be drawn, and the second period of the project will be dedicated to answering the following remaining major questions: [1] Is sulfur binding a general property of LbpA proteins from sulfur oxidizers? [2] What is the sulfur binding site in LbpA proteins? [3] Is sulfur binding the only function of LbpA proteins or does lipoate additionally switch between oxidized and reduced forms during the catalytic cycle of the Hdr-like complex, so that at least part of the electrons released during Hdr-driven sulfur oxidation can be used directly for the generation of NADH? [4] Are other sulfur transferases involved in the overall process? Answers to the above questions will be obtained by a combination of different experimental approaches. Genetic studies will include phenotypic characterization of H. denitrificans strains containing LbpA proteins with informative amino acid substitutions. Tagged LbpA and variants thereof will be purified and analyzed from cells grown in the absence/presence of oxidizable sulfur. In addition, we will focus on isolation of LbpA proteins from lithoautrophic sulfur oxidizers and enzyme assays with the sHdr proteins.

• DFG Programme: Research Grants