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Introduction
Basic Energy Concepts
Enzymes
Types of Catabolism
Fermentation
Feremented Foods
Respiration
Catabolism of Fats
Catabolism of Proteins
Amazing Respirations
Membranes and
Energy Generation

Anaerobic Respiration
Lithotrophs
Photosynthesis
Summary of Catabolism
Anabolism
Collecting Elements
Synthesizing Monomers
Carbon Assimilation
Nitrogen Assimulation
Other Assimilation
Formation of
Amino Acids

Lipid Synthesis
Nucleotide Synthesis
Making Polymers
Structural Assembly
Amphibolic Pathways

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Nitrogen Assimilation

©2000 Timothy Paustian, University of Wisconsin-Madison

Nitrogen is needed mostly for the synthesis of amino acids and nucleotides. Sources of nitrogen can be obtained from organic as well as inorganic sources, but the overall goal is the same; move it into the cell and then convert it to ammonia and amino acids.

Organic sources

Proteins available in the environment are acted upon by proteases releasing peptides that are further broken down to amino acids. The amino acids are then transported into the cell. These amino acids can be used directly for protein synthesis and some also contribute to nucleotide biosynthesis. Other nitrogen containing organic sources (urea, nucleotides or amines not used in protein synthesis) can be acted upon by enzymes (deaminases, decarboxylases and aminotransferases) to release the ammonia and make it available for biosynthesis.

Nitrate Reduction

Inorganic forms of nitrogen can also serve as sources of nitrogen. One of the most common compounds utilized is nitrate. Many bacteria, plants and fungi are capable of this assimilatory nitrate reduction. Nitrate is reduced to nitrite by the enzyme nitrate reductase and nitrite is then reduced to ammonia in a series of two electron transfers by nitrite reductase. The final product being ammonia which is readily incorporated into amino acids.

Figure 1 - Nitrate assimilation.

Nitrogen fixation

Nitrate in the natural environment is relatively rare. Microbes capable of using alternative nitrogen sources have an advantage and a subset of microbes is capable of obtaining the nitrogen they need from nitrogen gas. Nitrogen gas makes up about 79% of our atmosphere and is easily available. Molecular nitrogen is a stable unreactive gas with a triple bond between the two atoms and the reduction of it to ammonia is an energy expensive process. A large amount of ATP, protons and electrons are required to reduce just one molecule of nitrogen gas.

N2 + 8H+ + 8e- + 16ATP2 NH3 + H2 + 16ADP + 16Pi

Figure 2 - Chemical equation for the reduction of nitrogen gas to ammonia by nitrogenase.

The enzyme that catalyzes the reaction is called nitrogenase and is made of two separate protein components, dinitrogenase reductase and dinitrogenase. Dinitrogenase reductase prepares and donates two high potential electrons at a time to dinitrogenase. It contains an Fe-S center that holds the electrons before donation. Dinitrogenase actually catalyzes the reduction of N2. The mechanism of reduction is unknown but is thought to involve three 2e- transfers to nitrogen. Formation of hydrogen gas always accompanies the formation of ammonia by dinitrogenase and is a wasteful process. Some microorganisms have hydrogenases that recover this lost energy. Recently the complete crystal structure for dinitrogenase was solved. This was a herculean effort considering that dinitrogenase is a very large enzyme complex (240,000 daltons). Knowledge of the structure of dinitrogenase should lead to better understanding of the mechanism of nitrogen reduction on the enzyme.

Associations

Nitrogen fixation is important to humans for two reasons. A better understanding of the biological process which takes place at pH 7.0 at around 37°C could lead to better chemical synthesis methods than those currently employed that require high temperatures and pressures. About 1 % of the energy generated by humans is used to create nitrogen fertilizers; typically in the form of nitrate. Second, some nitrogen fixing microbes form cooperative (symbiotic) relationships with plants. The microbe fixes nitrogen for the plant and the plant provides carbon and energy for the microbes. Plants that are capable of forming these symbiotic relationships are called legumes. Many legumes are important crops including soy beans, peas and beans. A better understanding of the symbiosis and the biochemistry of nitrogen fixation should lead to improved crops and less need for nitrogen fertilizers that pollute our environment.

Regulation

Nitrogen fixation is costly to the microbe and because of this, its expression and activity are under tight control. In the presence of fixed forms of nitrogen, ammonia or nitrate, synthesis of the nitrogenase gene products, dinitrogenase and dinitrogenase reductase, is rapidly shut off. In some species, dinitrogenase reductase is covalently modified by the addition of an ADP-ribosyl unit. Professor Paul Ludden of the University of Wisconsin studies this process.

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