Elucidating the Origin and Evolution of Nitrogen Fixation by Resurrecting Ancestral Nitrogenase Enzymes

• Applicant: Johannes Rebelein, Ph.D.
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Biochemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 547781599

In this project, I aim to elucidate how the essential nitrogen fixation arose and evolved in nature. Although all forms of life require bioavailable nitrogen to build central metabolites, including nucleotides and amino acids, it appears that nature has solved the N-fixation problem only once during evolution. Nitrogenases are the only known enzymes able to break the inert triple bond of molecular nitrogen (N2) to produce bioavailable nitrogen (ammonia). Here, we will retrace the origin of nitrogen fixation by resurrecting nitrogenases from the past using ancestral sequence resurrection. In fact, we will revive the last common ancestor of the oldest nitrogenase, the molybdenum (Mo) nitrogenase, and the last common ancestor of the nitrogenase maturases to identify amino acids essential for catalysis and maturation. Subsequently, we will turn the predecessor into a catalytic Mo nitrogenase and a specific maturase. Turning a non-nitrogenase enzyme into a bona fide nitrogenase will provide new insights into the debated nitrogenase mechanism. Moreover, we will answer questions such as: i) How did the metallocluster composition and the active site of the nitrogenase predecessor look? ii) How does the first nitrogenase compare to the nitrogenase predecessor iii) What amino acid mutations turn a non-nitrogenase enzyme into a bona fide nitrogenase or a specific maturase? iv) What are the determining structural principles of nitrogenases and nitrogenase maturases? The proposed research will cast light on one of the most consequential events in biological history: the evolution of nitrogenases from non-nitrogen-fixing enzymes. To address these questions, we will use our established plasmid-based nitrogenase expression system in Rhodobacter capsulatus to express, screen and purify the oxygen-sensitive ancestral nitrogenases. The nitrogenases will be characterized catalytically by gas chromatography and photometric assays, structurally by single-particle cryogenic electron microscopy and crystallography, and spectroscopically by electron paramagnetic resonance spectroscopy to gain a comprehensive understanding of these ancestral enzymes. With these studies, we will gain first insights into the evolution of these highly reactive and complex metalloenzymes. We will unravel the structure and activities of different ancestral active site metallocofactors as well as the role of individual amino acids in nitrogen fixation and cluster maturation to gain a thorough understanding of the structure-function relationship. Through these studies, we will gain novel insights into the debated nitrogenase mechanism and potentially answer the question why nature solved the nitrogen fixation problem only once during evolution.

• DFG Programme: Research Grants