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Inorganic Nitrogen Assimilation

Inorganic Nitrogen Assimilation

Nitrogen is an essential element in biological systems, forming a crucial component of proteins, nucleic acids, and other cellular constituents. Many bacteria and archaea acquire nitrogen in the form of nitrate (NO₃⁻) or ammonia (NH₃), which are then assimilated into biomolecules through specific enzymatic pathways.Assimilatory Nitrate ReductionWhen nitrate enters the cell, it undergoes a two-step reduction process known as assimilatory nitrate reduction. Initially, the enzyme...

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A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation.

Lucía Payá-Tormo¹, Diana Coroian¹, Silvia Martín-Muñoz^1,2

¹Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Campus de Montegancedo UPM, Crta M-40 km 38 Pozuelo de Alarcón, 28223, Madrid, Spain.

Scientific Reports

|June 21, 2022

Summary

Researchers developed a new assay to screen nitrogenase components for engineering crops that can fix nitrogen. This method identified NifH proteins with NifM independency and oxygen resistance, crucial for sustainable agriculture.

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Area of Science:

Biochemistry
Molecular Biology
Agricultural Science

Background:

Biological nitrogen fixation (BNF) converts atmospheric nitrogen (N2) to ammonia (NH3) using oxygen-sensitive nitrogenase enzymes in prokaryotes.
Engineering crops for direct BNF could reduce synthetic fertilizer reliance and boost agricultural sustainability.
Current acetylene reduction assay (ARA) for nitrogenase activity is not suitable for high-throughput screening or automation.

Purpose of the Study:

To develop a microplate-based assay for high-throughput screening of nitrogenase components.
To identify NifH proteins with desirable traits for engineering nitrogen fixation in plants.
To assess NifH proteins for NifM independency and oxygen (O2) resistance.

Main Methods:

Developed and validated a reduced sulfonated viologen derivative (S2Vred) assay for measuring nitrogenase activity using a microplate reader.
Screened a library of NifH proteins targeted to yeast mitochondria.
Evaluated NifH proteins for NifM dependency, O2 tolerance, solubility, and [Fe-S] cluster occupancy.

Main Results:

The S2Vred assay successfully replaced the ARA for simultaneous analysis of isolated nitrogenase proteins.
Identified NifH from *Dehalococcoides ethenogenes* as NifM-independent, requiring fewer genes for transfer to eukaryotes.
Identified NifH from *Geobacter sulfurreducens* as exhibiting enhanced O2 resistance, crucial for stability in eukaryotic cells.

Conclusions:

The S2Vred assay is a viable, high-throughput alternative to ARA for nitrogenase activity screening.
Specific NifH proteins possess advantageous biochemical properties (NifM independency, O2 tolerance) for engineering BNF in plants.
Focusing on identifying such naturally occurring nitrogenase components is key for developing self-fertilizing crops.