Future directions in cofactor research: Discovery and development of coenzyme F420-dependent bioprocesses
Metabolism, Biochemistry and Genetics of Microorganisms
Final Report Abstract
Coenzyme F420 is a redox cofactor involved in specialized biochemical processes of microorganisms like methanogenesis, antibiotics biosynthesis, or drug resistance. Furthermore, F420-dependent enzymes have attracted increasing interest for their use in biocatalysis. The main objectives of this project were to identify and characterize novel deazaflavin-dependent enzymes/processes and facilitate biotechnological production and regeneration of the cofactor. During the project, we serendipitously discovered a novel derivative of coenzyme F420, 3PG- F420, from the symbiotic bacterium Mycetohabitans rhizoxinica that lives associated with phytopathogenic fungi. We elucidated the unknown molecule's structure and showed that it resembled classical F420 in its biochemical properties. Furthermore, we shed light on the biosynthesis and evolution of the novel cofactor derivative. We extensively investigated the roles of the biosynthetic enzymes CofC and CofD in the diversification of coenzyme F420 biosynthesis. Together with the group of M. Lammers (Greifswald) we obtained a crystal structure of CofC from Mycetohabitans sp. B3 and could clarify how biosynthesis switched from classical F420 to 3PG-F420 during evolution. We also discovered an important function of CofD in substrate selection. During these investigations, we also managed to clarify an essential step in the biosynthesis of coenzyme F420 in thermophilic bacteria. In addition, we succeeded in producing 3PG-F420 and F420 in the model organism E. coli and added a working regeneration system to the same system. Thus, we established an easy-touse platform to perform F420-dependent reactions in E. coli. While attempts to improve an isopropanol-based regeneration system to regenerate reduced F420H2 from F420 remained unsuccessful, we discovered further enzymes that are promising for that purpose. Last but not least, we made progress in the investigation of the role of 3PG-F420 in the symbiosis between bacteria of the genus Mycetohabitans and their fungal host Rhizopus microsporus. In summary, the funded project has brought to light numerous new findings in the field of deazaflavin cofactor research. Especially the discovery of 3PG-F420 opened an exciting field of research. Significant progress was also made in the areas of F420 biosynthesis and production. The role of 3PG-F420 in bacterial-fungal symbiosis remains particularly interesting and promising for basic research into novel functions of unusual cofactors.
Publications
- (2019) Metabolic pathway rerouting in Paraburkholderia rhizoxinica evolved long-overlooked derivatives of coenzyme F420. ACS Chem Biol 14, 2088-2094
Braga, D., Last, D., Hasan, M., Guo, H., Leichnitz, D., Uzum, Z., Richter, I., Schalk, F., Beemelmanns, C., Hertweck, C., and Lackner, G.
(See online at https://doi.org/10.1021/acschembio.9b00605) - (2020) Redox coenzyme F420 biosynthesis in Thermomicrobia involves reduction by stand-alone nitroreductase superfamily enzymes. Appl Environ Microbiol 86
Braga, D., Hasan, M., Kröber, T., Last, D., and Lackner, G.
(See online at https://doi.org/10.1128/aem.00457-20) - (2022) Diversification by CofC and control by CofD govern biosynthesis and evolution of coenzyme F420 and its derivative 3PG-F420. mBio 13, e03501-03521
Hasan, M., Schulze, S., Berndt, L., Palm, G. J., Braga, D., Richter, I., Last, D., Lammers, M., and Lackner, G.
(See online at https://doi.org/10.1128/mbio.03501-21)
