User:Jrapp99/Nitrogen metabolic pathways

Nitrogen Flow through Metabolism Nitrogen flow through metabolic networks are significant to the production of proteins and other nitrogen components, which also provides insight into the process of how proteins are made and their functions. Nitrogen is cycled through plants, animals, and bacteria/fungi through their metabolisms. The process begins when the bacteria fixates the nitrogen in the air and forms it into nitrogen compounds (i.e. ammonia, urea, nitrates, etc...). These compounds can be absorbed by plants and fungi through the root nodules in which the nitrogen compounds are then used to make proteins and other amino acids that help provide energy. Table of Contents...

1.    Nitrogen in the Soil


 * Nitrogen Fixation (Bacteria)


 * a.     Nitrification (conversion of ammonium to nitrate)


 * b.    Ammonification


 * c.     Denitrification

2.    Nitrogen Assimilation


 * a.     Conversion of nitrate to ammonia


 * b.    Conversion of Ammonia to amino acids


 * c.     Assimilation in Biotechnology

3.    Nitrogen flow through Animals


 * a.   Conversion of ammonia to organic nitrogen


 * b.   Nitrogen in the Body


 * c.    Ammonia Poisoning


 * d.    Human disturbances of the nitrogen cycle

4.    References

Nitrogen Fixation

Nitrification

The two-step process that converts ammonium to nitrite and then to nitrate. Nitrosomonas are bacteria that convert the ammonium to nitrite. The next step involves a bacteria called Nitrobacter which converts the nitrite to nitrate. These two reactions are coupled and rapidly synthesize nitrate.[1]

NH4+ → NO2- → NO3-

The bacteria that are involved in nitrification are also called nitrifiers. Other nitrifiers include Nitrococcus and Nitrosococcus. These bacteria take energy from the nitrogen compounds that have been reduced and create organic compounds. The process needs oxygen in order to occur; therefore nitrification is an aerobic process. Some requirements for nitrification to occur include, “a long retention time, a low food to microorganism ratio, a high mean cell residence time, and adequate buffering.” [2]

The product nitrate is very soluble so once it is released into soil, it is easily absorbed by bacteria and plants. Then the bacteria and plants can convert the nitrate into amino acids that they need.

Some factors that influence the rate of nitrification are pH, temperature, oxygen concentration, and inhibiting substances.

pH: Nitrification also produces acid, which in turn lowers pH. This can reduce the growth rate of the bacteria that actuate nitrification. An ideal pH for the bacteria involved in nitrification is between 7.5 and 8.5, which are basic pH levels. However, nitrification can successfully occur with pH levels of 6.5 to 7.0. Once the pH drops below 6.0, nitrification can no longer occur. Temperature: If temperature is changed rapidly, the reaction has a slow adaptation period. It has been approximated that temperatures from 0-20 degrees C maximize growth rates. Oxygen Concentration: Having enough oxygen for the reaction to proceed is essential for nitrification. Inhibiting Substances: Nitrification can be inhibited by certain substances, such as metals. If more than one inhibitor interferes with the reaction, the extent of inhibition rises. [3]

Ammonification:

In ammonification, he nitrogen that exists mostly in the form of proteins from decomposed plant and animal tissue is converted back into ammonia. All microorganisms that are involved in the decay of dead organic matter carry out the process. When a plant or animal dies the bacteria in the soil are able to start to break down the proteins through ammonification. The protease of soil bacteria hydrolyze the amino acids. Then a process called deanimation where enzymes, called deaminases, remove the amino groups (NH2) on the amino acids, then producing ammonia (NH3). Ammonium ions (NH4+) are often a product because soon after they can dissolve into water that is already present in the soil.[4] The protelyic enzymes that are used in ammonification can be found in organisms such as Clostridium ,Micrococcus, Proteus.[5]

Alanine

CH3 CHNH2 COOH + 1/2 O2 -> C H3COCOOH + NH3 Alanine deaminase  Pyruvic acid               ammonia [5]

Denitrification:

Denitrification is the process of reducing nitrates to nitrous oxide to nitrogen gases (NO3- -> NO2- -> N2O -> N2) which are then released into the atmosphere. Alcaligenes, Bacillus, Paracoccus and Pseudomonas are the heterotrophic bacteria that break down the nitrogen in the soil.[6] Normally this process happens in anaerobic or anoxic soils where there are abundant sources of organic matter to provide energy for the metabolic process. [6] Denitrification occurs when oxygen in the soil has been exhausted and thus causing nitrate to be the primary source of oxygen for microorganisms.[7] The nitrogen gas escapes into the atmosphere, since nitrogen is a major component of air, there is no environmental concern for this process. Denitrification is maximized when the soil is warm and saturated for days at a time with a ph between 7.0 and 8.5.[7] From denitrification the nitrogen moves from the soil system to other metabolisms such as those in plants and animals. Nitrogen is lost from the soil, by leaching – where NO 3 – moves with water to the region below the roots of plants, denitrification, and volatilization – nitrogen transforming into nitrogen gas and being released into the atmosphere, crop removal, erosion and runoff. [6]

Nitrogen Assimilation

Conversion of Nitrate to Ammonia

“Nitrate and ammonia in soil are forms of inorganic nitrogen that can be assimilated in all plants”; the problem is that atmospheric nitrogen can not be assimilated in most of the plants. Only in leguminous plants, with the help of microbes, can nitrogen be assimilated. Plants need to find a way to convert atmospheric nitrogen into an organic form that could be used by all plants. This conversion starts by the conversion of nitrate to ammonia. The nitrate can be absorbed by plants, and by using nitrate, plants reductase and nitrite reductase are able to then convert the nitrate back to ammonium ions (NH4+) which can be used to build amino acids.

NO3 → NO2

NADH → NAD+

NO2 → NH4

Red Ferredoxin

Amino Acid Production

After converting nitrogen to a usable form, ammonia, plants can synthesize amino acids, the building blocks of proteins. These reactions take place in the cells of both leaves and roots[8]. The production of amino acids takes place in many steps; however, a major step is the process is reductive animation, the formation of glutamate from ammonia and α-Ketoglutarate[9]. The enzyme glutamate dehydrogenase catalyzes this reaction. The enzyme is located in the mitochondria of both leaf and root cells, which are the sites of this mechanism of glutamate synthesis[10]. The reaction is as follows:


 * NH4+ + α-Ketoglutarate + NADPH → Glutamate Dehydrogenase → Glutamate + NADP

Glutamate and ammonia can react to synthesize another amino acid, glutamine, in a reaction catalyzed by glutamine synthetase[11]. This enzyme is located in chloroplasts and the cytosol of root and leaf cells. Its abundance and low Km values suggest that this reaction is a major route of nitrogen assimilation[12]:


 * NH4+ + ATP + Glutamate → Glutamine Synthase → Glutamine + ADP + P

The synthesis of glutamate can also proceed from a reaction of glutamine and α-Ketoglutarate, catalyzed by glutamate synthase. The enzyme is found in the chloroplasts of leaves and the plastids of root cells [10]:


 * GLutamine + α-Ketoglutarate → Glutamate Synthase → 2 Glutamate

Assimilation in Biotechnology

Examining the assimilation of nitrogen in plants is beneficial for researchers looking to improve amino acid production from ammonium in situations of nitrogen limitation. The metabolic pathways involved reveal important data that can be adapted in the biological engineering of nitrogen metabolism[13]. The biotechnological workhorse, known as Corynebacterium glutamicum, is controlled by nitrogen metabolism, which makes it especially valuable to researchers [13]. C. glutamicum is a small, gram-positive bacterium that uses fermentative metabolism to break down carbohydrates and is used to produce amino acids and nucleotides[14]. Its properties and capabilities make it ideal for use in various biotechnological applications, like improving amino acid production in cells[15]. Ammonium is a preferred nitrogen source of C. glutamicum and is assimilated via the Glutamine Synthetase pathway. When across the cytoplasmic membrane, diffusion of ammonia promotes growth and amino acid production [13]. The enhancing of Glutamine Synthetase pathway activity has shown to be beneficial for L-glutamine production[16]. There are also claims that enhancing the Glutamine Synthetase pathway can effect production of L-arginine and L-lysine[17]

Nitrogen Flow in Animals

For animals, their source of nitrogen is obtained through the food chain. Animals may prey on plants or other animals that have eaten plants and so forth. After plants obtain nitrogen, they maintain it in the form of ammonium. However, once animals feed on this nitrogen their bodies form organic nitrogen[18]. This organic nitrogen is used in the body to form amino acids. These amino acids can be synthesized in the human body from carbohydrates, nucleic acid, or lipid intermediates and are called non-essential amino acids. Amino acids that cannot be synthesized by animals or humans are called essential amino acids and they are obtained through the ingestion of plants. The purpose of these essential amino acids is so that the body may produce proteins to continue on all regular bodily functions. In addition, organic nitrogen in the body also goes to producing nucleic acids. Nucleic acids combine to produce Ribonucleic Acid (RNA) and Deoxyribonucleic Acid (DNA). With RNA and DNA, the body has genetic material to send signals throughout the body to perform all necessary functions. These functions are then carried out by the corresponding protein assigned to the task [18]. Though nitrogen only makes up 3% of the human body, it is vital that it is present.

The Liver

The liver plays a major role in coordinating metabolism in the body. Accordingly, it keeps the nitrogen intake and excretion balanced in animals and humans. Both protein synthesis and non-essential amino acid synthesis occur in the liver. Non-Essential Amino Acids are formed in the liver through reductive animation, using the carbon and the amino nitrogen atoms from glutamate. This allows for the recycling of ammonia instead of its excretion in form of urea[19]. Like plants, the liver can also synthesize glutamine from glutamate as glutamine is used in the body to transport ammonia between organs.

Nitrogen Digestion

Nitrogen enters the lumen of the gastrointestinal tract in the form of digestive secretions such as bile, mucins, urea and other nitrogenous substances[20]. Then, the nitrogen secreted either moves from the lumen into the body or is lost in the feces. For the most part, it moves into the body, only a small amount of nitrogen is lost. The gastrointestinal tract also digests and absorbs proteins. These are first broken down through proteolysis into peptide chains with the help of the enzyme pepsin, which comes from the proenzyme pepsinogen, secreted by the stomach, reacting with the hydrochloric acid (HCl) present in the stomach. Then, pancreatic and intestinal proteases keep breaking down these chains into smaller ones through hydrolysis resulting in individual amino acids [20]. The small intestine then absorbs the amino acid molecules allowing the bloodstream to transport them throughout the rest of the body.

To keep the nitrogen in cycle, one must understand how the nitrogen exits the body. Once the organic nitrogen has been processed in the body, animals may expel waste in various forms of organic nitrogen such as uric acid (C5H4N4O3) or urea ((NH)2CO). Bacteria can then act on the waste, or even dead animal carcass, to perform ammonification to convert the organic nitrogen back to ammonia. Enzymes Involved:

GS: Gln Synthetase (Cytosolic & PLastid)

GOGAT: Glu 2-oxoglutarate aminotransferase (Ferredoxin & NADH dependent)

GDH: Glu Dehydrogenase:

Minor Role in ammonium assimilation.

Important in amino acid catabolism.

Ammonia Poisoning

When ammonia enters the body as a result of breathing, swallowing or skin contact, it reacts with water to produce ammonium hydroxide. This chemical is very corrosive and damages cells in the body on contact.[22]

Human Alterations of the Nitrogen Cycle

The balance of the nitrogen cycle is extremely important to maintain. However, due to humans immensely industrializing nitrogen fixation, the amount of available nitrogen on the planet has increased exponentially. This is extremely dangerous because this directly affects both terrestrial and aquatic ecosystems [21]. The most significant way that nitrogen harms the environment is through the Haber-Bosch process. This is where chemically produced nitrogen is saturated in fertilizer to help increase yield of crops. With extra nitrogen not absorbed by the soil, the nitrogen flows in runoff into lakes and rivers. From there, the reactive nitrogen, such as nitrates and ammonium react with the water. In result, nitric oxide (NO) and the greenhouse gas, nitrous oxide (N20) are formed that accumulates in the atmosphere [18]. This causes decreases of biodiversity to the planet as a whole.