Citrulline participates in various key physiological functions within living organisms. Although it does not directly engage in regular protein synthesis, it is found in some specialized proteins, such as myelin basic protein in the human brain. Citrulline is converted into nitric oxide, which is involved in physiological processes like vasodilation and regulates cell signaling. Abnormalities in citrulline metabolism are associated with multiple diseases, including hypertension and atherosclerosis in cardiovascular diseases, as well as neurodegenerative disorders like Alzheimer's and Parkinson's diseases. Therefore, in-depth research on citrulline metabolism holds significant potential for uncovering disease mechanisms, developing diagnostic markers, and formulating targeted therapeutic strategies.
Biosynthesis of citrulline
Citrulline was first isolated from watermelon juice by Japanese scientists in 1914 and named after its source. In nature, citrulline is widely distributed. Watermelon is a typical plant rich in citrulline, and certain amounts of citrulline are also found in other cucurbitaceous plants such as pumpkins and cucumbers. In animals, citrulline is primarily produced through the metabolism of intestinal cells and is present in organs such as the liver and kidneys. Additionally, some bacteria and fungi synthesize and utilize citrulline during specific metabolic processes, indicating its diverse functions in biological activities.
Enzymatic Reaction Mechanism
The biosynthesis of citrulline involves multiple key enzymes, among which argininosuccinate synthetase plays a crucial role. The active site of this enzyme has a unique three-dimensional structure formed by specific amino acid residues, creating a precise microenvironment for substrate binding and catalysis. The substrate binding mechanism exhibits high specificity, allowing the enzyme to selectively recognize and bind aspartate and citrulline. This interaction follows an induced-fit model, ensuring optimal alignment between the substrates and the enzyme's active site.
During the catalytic process, an intermediate, argininosuccinate, is first formed, a step that involves energy consumption and chemical bond rearrangement. Subsequently, argininosuccinate is cleaved by argininosuccinate lyase, yielding citrulline and fumarate as final products.
In addition to argininosuccinate synthetase, ornithine transcarbamylase also plays a pivotal role in citrulline biosynthesis. The enzymes involved in this pathway function in a highly coordinated manner, sequentially transforming precursor molecules into citrulline in a relay-like fashion. This enzymatic synergy ensures the efficiency and precision of citrulline biosynthesis, maintaining a stable supply within the organism.
Figure 1. Citrulline metabolism pathways.
Major Metabolic Pathways
Citrulline biosynthesis is a critical step in the urea cycle. Initially, ammonia and carbon dioxide are catalyzed by carbamoyl phosphate synthetase, consuming ATP to form carbamoyl phosphate, which serves as the primary substrate for subsequent citrulline synthesis. Next, carbamoyl phosphate reacts with ornithine under the catalysis of ornithine transcarbamylase to produce citrulline. This highlights the essential role of ammonia and carbon dioxide as starting materials in providing the necessary elemental foundation for citrulline synthesis.
The downstream processes of the urea cycle are closely linked to citrulline. Citrulline combines with aspartate in a reaction catalyzed by argininosuccinate synthetase to form argininosuccinate, which is then cleaved by argininosuccinate lyase into arginine and fumarate. Arginine is subsequently hydrolyzed by arginase to produce urea and ornithine, the latter re-entering the cycle to participate in further citrulline synthesis. This orderly progression of the urea cycle ensures nitrogen homeostasis in the body, with citrulline serving as a crucial intermediary.
Alternative Metabolic Pathways
Certain microorganisms have unique pathways for citrulline biosynthesis that differ from the conventional urea cycle. For instance, some bacteria can synthesize citrulline through specialized enzymatic systems using alternative nitrogen and carbon sources from their environment.
Compared to the classical urea cycle, microbial citrulline biosynthesis exhibits distinct characteristics. These organisms may utilize uncommon precursor compounds instead of relying primarily on ammonia and carbon dioxide. Additionally, the types and catalytic mechanisms of the enzymes involved differ, as microbes have evolved unique enzyme systems adapted to their specific ecological niches. However, these alternative pathways share fundamental principles with the urea cycle, as they also involve sequential enzymatic reactions to produce citrulline. Ultimately, these pathways serve the common purpose of fulfilling the organism's metabolic requirements, maintaining homeostasis, and supporting growth and development.
Metabolic Transformation of Citrulline
Catabolic Pathway
Within cells, the degradation of citrulline is primarily catalyzed by citrullinase, an enzyme with high specificity that recognizes and binds citrulline. Through a hydrolytic reaction, citrullinase cleaves specific chemical bonds, breaking down citrulline into ornithine and urea. This process is highly precise and efficient, ensuring an orderly catabolism of citrulline. The ornithine produced can either enter the urea cycle or participate in other metabolic pathways, while urea is primarily excreted by the kidneys to maintain nitrogen homeostasis in the body.
Under different physiological conditions, citrulline catabolism exhibits significant variations. During starvation, when energy supply is insufficient, the catabolic rate of citrulline increases to meet energy demands. More citrulline is degraded into ornithine and urea, with ornithine further participating in gluconeogenesis to provide energy. This metabolic adaptation helps maintain energy balance during starvation.
During exercise, muscle metabolism intensifies, increasing the demand for both energy and amino acids. Consequently, citrulline catabolism is also adjusted accordingly. On one hand, the degradation rate increases, supplying more ornithine and urea to support muscle metabolism. On the other hand, the metabolic fate of ornithine is altered—some ornithine is utilized for creatine synthesis, which enhances muscle strength and endurance, meeting the specific demands of muscle function during physical activity. These regulatory changes reflect the adaptive flexibility of citrulline catabolism, which plays a vital role in sustaining normal physiological functions.
Interconversion with Other Amino Acids
Citrulline has a close metabolic relationship with arginine and ornithine, undergoing interconversion through enzymatic reactions. Citrulline can be converted into arginine via the sequential actions of argininosuccinate synthetase and argininosuccinate lyase. This transformation requires energy and involves multiple enzymatic steps: first, citrulline reacts with aspartate under the catalysis of argininosuccinate synthetase to form argininosuccinate; then, argininosuccinate is cleaved by argininosuccinate lyase into arginine and fumarate.
These metabolic transformations are accompanied by energy fluctuations. The conversion of citrulline to arginine is an ATP-dependent process, requiring energy input to drive the reaction. In contrast, the breakdown of citrulline into ornithine is relatively energy-releasing, allowing the liberated energy to be utilized by the body.
In the broader amino acid metabolic network, citrulline occupies a pivotal position. As a central hub linking multiple amino acid metabolic pathways, citrulline plays a crucial role in maintaining amino acid homeostasis through its interconversion with arginine and ornithine. When the body's demand for a specific amino acid changes, citrulline serves as a metabolic regulator, ensuring a stable supply of amino acids. This regulatory function is essential for sustaining normal cellular physiology, protein synthesis, and overall metabolic balance.
Citrulline Metabolism in Different Biological Systems
Metabolic Characteristics in Mammals
In mammals, the liver and kidneys are the primary organs involved in citrulline metabolism, with different tissues contributing variably to its metabolic processes. In the liver, citrulline serves as an intermediate in the urea cycle. Within hepatocytes, ammonia and carbon dioxide are converted into citrulline through enzymatic reactions, which then continues through the urea cycle to produce urea for excretion. This process is essential for maintaining nitrogen homeostasis in the body.
The kidneys play another critical role in citrulline metabolism by converting circulating citrulline into arginine. Proximal tubule cells in the kidneys express specific enzymes that efficiently catalyze this conversion, releasing arginine into the bloodstream to supply other tissues and organs.
Citrulline metabolism varies across different physiological stages in mammals. During embryonic development, citrulline is closely linked to growth and differentiation, participating in various biosynthetic pathways necessary for organ formation. In adulthood, citrulline metabolism stabilizes, primarily supporting physiological functions such as vascular regulation and immune responses. However, in aging, metabolic alterations occur due to declining enzymatic activity, leading to reduced citrulline synthesis and conversion efficiency. This imbalance in nitrogen metabolism may contribute to age-related diseases. Understanding these metabolic shifts across life stages is crucial for ensuring proper development and health in mammals.
Metabolic Characteristics in Plants
Citrulline plays a vital role in various physiological processes in plants, particularly in energy metabolism and nitrogen assimilation. During photosynthesis, citrulline may act as an intermediate metabolite in the dark reaction, contributing to carbon fixation and organic compound synthesis, which provides essential energy and structural components for plant growth. In nitrogen assimilation, citrulline is a key intermediate in the conversion of inorganic nitrogen from the soil into amino acids and proteins, supporting normal growth and development.
Figure 2. Nitrogen uptake, assimilation in roots and leaves of tea plant. (Li, W., et al., 2017)
When plants encounter environmental stressors such as drought, salinity, or high temperatures, citrulline metabolism undergoes adaptive changes. Under drought conditions, citrulline levels may increase to regulate cellular osmotic pressure, helping retain water and enhance drought tolerance. In saline-alkaline environments, citrulline metabolism aids in mitigating ion toxicity and maintaining intracellular ionic balance. Under heat stress, citrulline may be involved in antioxidant defense mechanisms, scavenging reactive oxygen species (ROS) to minimize oxidative damage. These adaptive responses highlight the physiological importance of citrulline metabolism in plant survival under adverse environmental conditions.
Metabolic Characteristics in Microorganisms
Microorganisms exhibit unique metabolic strategies for citrulline utilization. Bacteria and yeast can use citrulline as both a carbon and nitrogen source to sustain their growth and proliferation. Some bacterial species possess specialized transport proteins that facilitate citrulline uptake from the environment. Once inside the cell, citrulline undergoes enzymatic degradation and transformation through metabolic pathways that supply energy and biosynthetic precursors. The carbon backbone and nitrogen atoms of citrulline are efficiently integrated into microbial metabolism, fulfilling essential cellular requirements.
Citrulline metabolism in microorganisms has significant applications in biotechnology. In industrial biosynthesis, microbial metabolic pathways can be harnessed to produce valuable biochemicals, including amino acids and vitamins. Through genetic engineering, microbial strains can be optimized to enhance citrulline metabolic flux, improving the yield and quality of desired products. Additionally, in bioremediation, certain microorganisms utilize citrulline metabolism to degrade environmental pollutants. Some bacteria, when exposed to citrulline, exhibit enhanced degradation of organic contaminants, converting them into non-toxic substances and contributing to environmental cleanup.
In-depth research into microbial citrulline metabolism holds great potential for advancing biotechnology and environmental science, paving the way for innovative solutions in sustainable production and ecological restoration.
Citrulline Metabolism and Its Relationship with Diseases
Association with Cardiovascular Diseases
Multiple studies have demonstrated a close link between aberrant citrulline metabolism and cardiovascular diseases. In hypertensive patients, citrulline levels may be altered. Research has found that plasma citrulline concentrations in hypertensive individuals are lower than those in healthy populations, potentially due to the activation of the renin-angiotensin system, which affects citrulline synthesis or metabolic pathways. Additionally, citrulline metabolism plays a crucial role in atherosclerosis. Nitric oxide (NO), a key product of citrulline metabolism, exerts vasodilatory effects and inhibits platelet aggregation. When citrulline metabolism is disrupted, NO production decreases, leading to endothelial dysfunction and promoting lipid deposition on arterial walls, thereby accelerating the progression of atherosclerosis.
Given these findings, citrulline and its metabolites may serve as biomarkers for cardiovascular diseases. Plasma citrulline level assessments can aid in the early detection of cardiovascular disease risks and provide dynamic monitoring for disease progression and prognosis. Modulating citrulline metabolism has also emerged as a potential therapeutic strategy. For example, citrulline supplementation or enzymatic activity regulation may help restore endothelial function and reduce the risk of cardiovascular disease. Ongoing clinical trials are investigating dietary and pharmacological interventions targeting citrulline metabolism, offering new insights into cardiovascular disease prevention and treatment.
Association with Neurological Disorders
Citrulline plays a vital physiological role in the nervous system. It is involved in neurotransmitter synthesis and regulation, ensuring normal neuronal function and synaptic transmission. Additionally, NO, a product of citrulline metabolism, contributes to neuroplasticity and neuroprotection.
However, disruptions in citrulline metabolism have been linked to neurodegenerative diseases. In Alzheimer's disease, studies have reported altered enzymatic activity associated with citrulline metabolism in the brain, leading to the accumulation of abnormal metabolites. These metabolic imbalances may exert neurotoxic effects, damaging neuronal structure and function and accelerating neuronal degeneration. Similarly, in Parkinson's disease, citrulline metabolic dysregulation may impair dopaminergic neurons, reducing dopamine secretion and contributing to motor dysfunction.
Further investigations have revealed that mutations in citrulline metabolism-related genes are closely associated with the pathogenesis of neurological disorders. Specific gene mutations may lead to structural and functional abnormalities in citrulline metabolic enzymes, disrupting normal metabolic pathways. Targeting citrulline metabolism for neurological disease treatment has thus become a promising therapeutic approach. Potential strategies include developing drugs that modulate citrulline metabolic enzyme activity or employing gene therapy to correct pathogenic mutations, offering new hope for neurological disorder treatment.
Association with Other Diseases
Citrulline metabolism is also closely associated with immune system disorders and metabolic syndromes. In immune-related diseases such as rheumatoid arthritis, inflammatory responses lead to amino acid metabolic imbalances, potentially altering citrulline levels. Abnormal citrulline metabolism may affect immune cell function and inflammatory cytokine release, exacerbating inflammation.
Citrulline metabolism offers multiple therapeutic targets for disease treatment. In immune-related disorders, modulating citrulline metabolism could help suppress excessive inflammatory responses. For metabolic syndrome, regulating citrulline metabolism may improve insulin resistance and restore energy homeostasis. Potential therapeutic approaches include developing drugs targeting key enzymes in citrulline metabolism or using dietary and lifestyle interventions to maintain metabolic balance, providing novel strategies for disease management.
Figure 3. Ass1-mediated citrulline consumption directly regulates proinflammatory activation and immune function of macrophages. (Mao, Y., et al., 2022)
Conclusion
Citrulline metabolism plays a crucial role in various physiological processes, from nitrogen homeostasis and cardiovascular regulation to neurological function and stress adaptation. Its metabolic pathways are intricately linked to multiple biological systems, with significant implications for health and disease. Understanding the enzymatic mechanisms, metabolic transformations, and interorgan relationships of citrulline metabolism provides valuable insights into potential therapeutic targets. As research continues to uncover new aspects of citrulline's biological significance, its applications in medicine, biotechnology, and nutrition are expected to expand, offering new avenues for disease prevention and treatment.
References
- Li, W., Xiang, F., Zhong, M., Zhou, L., Liu, H., Li, S., & Wang, X. (2017). Transcriptome and metabolite analysis identifies nitrogen utilization genes in tea plant (Camellia sinensis). Scientific reports, 7(1), 1693. https://doi.org/10.1038/s41598-017-01949-0
- Mao, Y., Shi, D., Li, G., & Jiang, P. (2022). Citrulline depletion by ASS1 is required for proinflammatory macrophage activation and immune responses. Molecular cell, 82(3), 527–541.e7. https://doi.org/10.1016/j.molcel.2021.12.006
