Novel lipoate-binding proteins and their role in sulfur oxidation

• Applicant: Privatdozentin Dr. Christiane Dahl
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433613342

Lipoic acid (1,2-dithiolane-3-pentanoic acid) is a highly conserved organosulfur cofactor found in almost all prokaryotic and eukaroytic organisms. The cofactor is essential for the function of several key enzymes involved in oxidative and one-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.Although the existence of lipoic acid has been known for more than sixty years, our recent work revealed an unexpected metabolic function for this cofactor that is markedly different from its canonical roles in central metabolism. We proved that a novel lipoate-binding proteins (LbpA) act as indispensable components of a new pathway of dissimilatory sulfur oxidation. A heterodisulfide reductase (Hdr)-like complex is a central player in this pathway. It occurs in a huge organism group that includes biotechnologically and environmentally relevant bacteria like the volatile organic sulfur compound degrading Alphaproteobacerium Hyphomicrobium denitrificans and many chemolithoautotrophic bacteria and archaea. In all cases known so far, lipoate acts as a swinging arm that channels bound substrate between the active sites of different subunits. During catalysis, the intramolecular disulfide bond of lipoate cycles between oxidized lipoamide and reduced dihydrolipoamide and the electrons released upon reoxidation of dihydroliponamide can be directly transferred onto NAD+ via dihydroliponamide dehydrogenase. If lipoate-binding proteins perform similar functions in Hdr-dependent sulfur oxidation, then at least some of the electrons released here could be used directly for the formation of NADH. Such a reaction would considerably reduce the need for energy-demanding reverse electron flow in sulfur-oxidizing lithoautotrophs. Direct biochemical or genetic evidence for these exciting suggestions is currently not available. In this project, we aim at at filling this gap and intend to clarify the exact role of the novel lipoate binding proteins during sulfur oxidation. The following major questions will be addressed: [1] Do LbpA proteins function as sulfur-binding entities presenting substrate to different catalytic sites of the new heterodisulfide reductase-like sulfur-oxidizing complex? [2] Does lipoate switch between its oxidized and reduced forms during the catalytic cycle of the Hdr-like complex such that at least part of the electrons released during Hdr-driven sulfur oxidation can be directly used for generation of NADH? [3] Which other sulfur transferases are involved in the process?

• DFG Programme: Research Grants