Asparagine synthetase

Last updated
asparagine synthetase
PDB 11as EBI.jpg
Identifiers
SymbolASNS
Alt. symbols11as, AsnS
NCBI gene 440
HGNC 753
OMIM 108370
RefSeq NM_001673
UniProt P08243
Other data
EC number 6.3.5.4
Locus Chr. 7 q21-q31
Search for
Structures Swiss-model
Domains InterPro

Asparagine synthetase (or aspartate-ammonia ligase) is a chiefly cytoplasmic enzyme that generates asparagine from aspartate. [1] This amidation reaction is similar to that promoted by glutamine synthetase. The enzyme is ubiquitous in its distribution in mammalian organs, but basal expression is relatively low in tissues other than the exocrine pancreas. [2]

Contents

Above average presence of asparagine synthetase in certain leukemia strains has been linked to be a significant contributing factor of chemotherapy resistance, particularly to the chemotherapy drug, L-asparaginase. [3]

Structure

Escherichia coli derived asparagine synthetase is a dimeric protein with each subunit folding into two distinct domains. [4] The N-terminal region consists of two layers of six-stranded antiparallel β-sheets between which is the active site responsible for the hydrolysis of glutamine. [4] The C-terminal domain consists of a five-stranded parallel β-sheet flanked on either side by α-helices. This domain is responsible for the binding of both Mg2+ATP and aspartate. [4] These two active sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic, nonpolar amino acid residues. [4]

Structural characterization of asparagine synthetase from mammalian sources have been difficult due to the low abundance and instability of the enzyme during purification procedures. [5]

Mechanism

Using information from Escherichia coli derived asparagine synthetase, some basic mechanisms of the enzyme have been understood. [5] The N-terminal active site catalyzes glutamine hydrolysis to yield glutamate and ammonia. [5] The C-terminal active site catalyzes activation of the side-chain carboxylate of aspartate to form an electrophilic intermediate, β-aspartyl-AMP (βAspAMP) 1, and inorganic pyrophosphate (PPi). [5] The tunnel that links the two active sites allows for the passage of an ammonia molecule to act as a common intermediate to couple the two half-reactions carried out in the independent active sites of the enzyme. [5] Thus, after being released in, and channeled from, the glutaminase site, the ammonia molecule attacks the bound βAspAMP 1 to give asparagine and AMP via a tetrahedral intermediate. [5]

Function

In plants, inorganic nitrogen is taken up from the environment in forms of nitrate or ammonium. [6] Assimilation of this nitrogen into asparagine for use in nitrogen recycling, transport, and storage is an essential process for plant development, making asparagine synthetase vital to maintaining these asparagine reserves. [6] Specific events in development which depend on asparagine synthetase are nitrogen mobilization in germinating seeds, nitrogen recycling and flow in vegetative cells in response to biotic and abiotic stresses, and nitrogen remobilization from source to sink organs. [6]

In mammals, asparagine synthetase expression has been found to be linked to cell growth, and its mRNA content is linked to changes in the cell cycle. [5] Hamster BHK ts11 cells produce an inactive asparagine synthetase enzyme, and this loss of asparagine synthetase activity directly led to cell cycle arrest in the cells as a consequence of a depletion of cellular asparagine. [5] Upregulation of asparagine synthetase mRNA was observed as well in these hamster cells. [5] Other experiments demonstrated that quiescent rat thyroid cells entering S phase as a result of thyroid-stimulating hormone treatment was matched with a concurrent increase in asparagine synthetase mRNA content. [5]

Classes

There seem to be two major groups of asparagine synthetase: [7] [6]

Clinical significance

Cancer

Leukemia

Cancerous cells exhibit rapid growth and cell division and subsequently have an increased nutritional need. [5] The particularly low-level expression of asparagine synthetase in primary acute lymphoblastic leukemia (ALL) and numerous ALL cell lines, as compared to that of normal cells, makes asparagine depletion an effective method of treatment due to the cells' unusual dependency on circulating serum asparagine as a necessary nutrition for growth. [2] [5] As a result, L-asparaginase is a common chemotherapy drug utilized in the treatment of ALL and may have applications in other asparagine synthetase negative cancers, such as lymphomas, due to its aspariginase activity to deplete serum asparagine. [9] This depletion of serum asparagine leads to a subsequent rapid efflux of cellular asparagine, which is immediately acted upon and destroyed by the L-asparaginase as well. [5] Due to the transient response from these susceptible cancers in reaction to the asparagine depletion, tumor growth is significantly inhibited due to nutritional deficiency. [5] [3]

Most somatic cells express sufficient basal amounts of asparagine synthetase to counteract this asparagine starvation and survive the effects of L-asparaginase. [2] [3] [5] In addition, these normal cells are able to upregulate their expression of asparagine synthetase in response to the asparagine depletion, further countering some of the toxic effects of the medication on normal cell activity, a desirable trait for chemotherapy drugs. [2] [3] [5]

However, the opposite effect is visible in cases of asparaginase resistant cancers. [3] In these resistant cancers, the effect of blood asparagine depletion through L-asparaginase instead leads to significant asparagine synthetase overexpression to compensate, effectively nullifying the effect of the chemotherapy drug. [3] For example, in mouse models, 24 hours after exposure to L-asparaginase, tumors resistant to the depletion responded with 5- to 19-fold increases in asparagine synthetase expression. [10] These resistant tumors also inherently express higher levels of asparagine synthetase activity, even without the application of L-asparaginase to drive further expression. [11] Similar trends are often seen in human studies as well, with high levels of asparagine synthetase activity being detected in asparaginase-resistant cases of treatment as compared with the negligible activity of susceptible cases. [12] As seen in in vitro studies of resistant human leukemia cell lines, even six weeks after the removal of asparagine depleting factors, the increased level of expression of asparagine synthetase failed to return to a basal state, instead remaining elevated and retaining continued drug resistance. [13]

While the mechanisms underlying the sustained over-expression of ASNS have not been reported in these studies, transcriptome profiling of two T-ALL patients that have relapsed after L-asparaginase treatment revealed a recurrent promoter swap with KMT2E leading to ASNS over-expression and L-asparaginase resistance. [14] It has been further demonstrated in mouse model systems that repeated subculturing of L-asparaginase sensitive tumor cells in sublethal concentrations of L-asparaginase could eventually make them resistant, a potential worry of lower dose chemotherapy treatments effectively encouraging resistant cell development. [15]

Potential biomarker for ovarian cancer

A correlation between L-asparaginase efficacy and asparagine synthetase protein levels in a number of human ovarian cell lines has been observed. [16] As mentioned above, this result confirmed similar observations in human leukemia cell lines. [16] Hence asparagine synthetase might be used as a biomarker in ovarian cancer screening and potential treatment. [16]

Potential role in solid tumor metastasis

An epithelial to mesenchymal transition was mimicked in metastatic cells by adapting PC-3 prostate cancer cells from adherent to suspension culture and then examined to investigate changes in gene expression concurrent with this adaption to suspension. [17] It was found that the asparagine synthetase expression was sixfold greater in the suspension cells than in the adherent cells. [17] In xenografts from a human breast cancer cell line in an established metastatic mouse model, [2] [18] asparagine synthetase was elevated in circulating tumor cells isolated from the mouse blood compared with the parental cell line. [2] [18] When these circulating tumor cells were returned to an in vitro culture and exposed to hypoxia, they showed higher basal expression and greater induction of asparagine synthetase than their parental cell line. [2] [18] These circulating tumor cells were also found to have an increased capacity for colony formation in soft agar assays under hypoxic conditions and grew faster when reimplanted as xenografts. [2] [18] The increased prevalence of asparaginase synthetase in the metastatic cells suggests that its activity may be beneficial for circulating tumor cell survival. [2] [18]

Trivia

Guinea pigs have some of the highest levels of naturally expressing asparagine synthetase due to the fact that their serum inherently containing detectable levels of L-asparaginase. [10]

Related Research Articles

<span class="mw-page-title-main">Asparagine</span> Chemical compound

Asparagine is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group, an α-carboxylic acid group, and a side chain carboxamide, classifying it as a polar, aliphatic amino acid. It is non-essential in humans, meaning the body can synthesize it. It is encoded by the codons AAU and AAC.

In molecular biology, biosynthesis is a multi-step, enzyme-catalyzed process where substrates are converted into more complex products in living organisms. In biosynthesis, simple compounds are modified, converted into other compounds, or joined to form macromolecules. This process often consists of metabolic pathways. Some of these biosynthetic pathways are located within a single cellular organelle, while others involve enzymes that are located within multiple cellular organelles. Examples of these biosynthetic pathways include the production of lipid membrane components and nucleotides. Biosynthesis is usually synonymous with anabolism.

<span class="mw-page-title-main">Glutamine synthetase</span> Class of enzymes

Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine:

<span class="mw-page-title-main">Asparaginase</span> Enzyme used as medication and in food manufacturing

Asparaginase is an enzyme that is used as a medication and in food manufacturing. As a medication, L-asparaginase is used to treat acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL). It is given by injection into a vein, muscle, or under the skin. A pegylated version is also available. In food manufacturing it is used to decrease acrylamide.

<span class="mw-page-title-main">Argininosuccinate synthase</span> Enzyme

Argininosuccinate synthase or synthetase is an enzyme that catalyzes the synthesis of argininosuccinate from citrulline and aspartate. In humans, argininosuccinate synthase is encoded by the ASS gene located on chromosome 9.

<span class="mw-page-title-main">Amino acid synthesis</span> The set of biochemical processes by which amino acids are produced

Amino acid synthesis is the set of biochemical processes by which the amino acids are produced. The substrates for these processes are various compounds in the organism's diet or growth media. Not all organisms are able to synthesize all amino acids. For example, humans can synthesize 11 of the 20 standard amino acids. These 11 are called the non-essential amino acids).

<span class="mw-page-title-main">Indoleamine 2,3-dioxygenase</span> Mammalian protein found in Homo sapiens

Indoleamine-pyrrole 2,3-dioxygenase (IDO or INDO EC 1.13.11.52) is a heme-containing enzyme physiologically expressed in a number of tissues and cells, such as the small intestine, lungs, female genital tract or placenta. In humans is encoded by the IDO1 gene. IDO is involved in tryptophan metabolism. It is one of three enzymes that catalyze the first and rate-limiting step in the kynurenine pathway, the O2-dependent oxidation of L-tryptophan to N-formylkynurenine, the others being indolamine-2,3-dioxygenase 2 (IDO2) and tryptophan 2,3-dioxygenase (TDO). IDO is an important part of the immune system and plays a part in natural defense against various pathogens. It is produced by the cells in response to inflammation and has an immunosuppressive function because of its ability to limit T-cell function and engage mechanisms of immune tolerance. Emerging evidence suggests that IDO becomes activated during tumor development, helping malignant cells escape eradication by the immune system. Expression of IDO has been described in a number of types of cancer, such as acute myeloid leukemia, ovarian cancer or colorectal cancer. IDO is part of the malignant transformation process and plays a key role in suppressing the anti-tumor immune response in the body, so inhibiting it could increase the effect of chemotherapy as well as other immunotherapeutic protocols. Furthermore, there is data implicating a role for IDO1 in the modulation of vascular tone in conditions of inflammation via a novel pathway involving singlet oxygen.

<span class="mw-page-title-main">CAD protein</span> Protein-coding gene in the species Homo sapiens

CAD protein is a trifunctional multi-domain enzyme involved in the first three steps of pyrimidine biosynthesis. De-novo synthesis starts with cytosolic carbamoylphosphate synthetase II which uses glutamine, carbon dioxide and ATP. This enzyme is inhibited by uridine triphosphate.

<span class="mw-page-title-main">CTP synthetase</span> Enzyme

CTP synthase is an enzyme involved in pyrimidine biosynthesis that interconverts UTP and CTP.

In enzymology, an asparaginyl-tRNA synthase (glutamine-hydrolysing) is an enzyme that catalyzes the chemical reaction

In enzymology, an aspartate—ammonia ligase (ADP-forming) (EC 6.3.1.4) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Omega-amidase</span>

In enzymology, an omega-amidase (EC 3.5.1.3) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Folylpolyglutamate synthase</span> Mammalian protein found in Homo sapiens

Folylpolyglutamate synthase, mitochondrial is an enzyme that in humans is encoded by the FPGS gene.

Glutaminolysis (glutamine + -lysis) is a series of biochemical reactions by which the amino acid glutamine is lysed to glutamate, aspartate, CO2, pyruvate, lactate, alanine and citrate.

6-Diazo-5-oxo-<small>L</small>-norleucine Chemical compound

6-Diazo-5-oxo-L-norleucine (DON) is a glutamine antagonist, which was isolated originally from Streptomyces in a sample of Peruvian soil. This diazo compound is biosynthesized from lysine by three enzymes in bacteria. It is one of the most famous non-proteinogenic amino acid and was characterized in 1956 by Henry W Dion et al., who suggested a possible use in cancer therapy. This antitumoral efficacy was confirmed in different animal models. DON was tested as chemotherapeutic agent in different clinical studies, but was never approved. In 2019, DON was shown to kill tumor cells while reversing disease symptoms and improve overall survival in late-stage experimental glioblastoma in mice, when combined with calorie-restricted ketogenic diet.

<span class="mw-page-title-main">CTP synthase 1</span> Protein-coding gene in the species Homo sapiens

CTP synthase 1 is an enzyme that is encoded by the CTPS1 gene in humans. CTP synthase 1 is an enzyme in the de novo pyrimidine synthesis pathway that catalyses the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP). CTP is a key building block for the production of DNA, RNA and some phospholipids.

<span class="mw-page-title-main">Purine nucleotide cycle</span>

The Purine Nucleotide Cycle is a metabolic pathway in protein metabolism requiring the amino acids aspartate and glutamate. The cycle is used to regulate the levels of adenine nucleotides, in which ammonia and fumarate are generated. AMP converts into IMP and the byproduct ammonia. IMP converts to S-AMP (adenylosuccinate), which then converts to AMP and the byproduct fumarate. The fumarate goes on to produce ATP (energy) via oxidative phosphorylation as it enters the Krebs cycle and then the electron transport chain. Lowenstein first described this pathway and outlined its importance in processes including amino acid catabolism and regulation of flux through glycolysis and the Krebs cycle.

<span class="mw-page-title-main">Asparagine synthase (glutamine-hydrolysing)</span>

Asparagine synthase (glutamine-hydrolysing) (EC 6.3.5.4, asparagine synthetase (glutamine-hydrolysing), glutamine-dependent asparagine synthetase, asparagine synthetase B, AS, AS-B) is an enzyme with systematic name L-aspartate:L-glutamine amido-ligase (AMP-forming). This enzyme catalyses the following chemical reaction

The glnALG operon is an operon that regulates the nitrogen content of a cell. It codes for the structural gene glnA the two regulatory genes glnL and glnG. glnA encodes glutamine synthetase, an enzyme which catalyzes the conversion of glutamate and ammonia to glutamine, thereby controlling the nitrogen level in the cell. glnG encodes NRI which regulates the expression of the glnALG operon at three promoters, which are glnAp1, glnAp2 located upstream of glnA) and glnLp. glnL encodes NRII which regulates the activity of NRI. No significant homology is found in Eukaryotes.

<span class="mw-page-title-main">Fructose-asparagine</span> Chemical compound

Fructose-asparagine (F-Asn) is a glycosylamine compound that is most notably used by Salmonella during Salmonella-mediated inflammation of the intestine. In addition to Salmonella, several other species of bacteria may utilize fructose-asparagine as a nutrient. The name of the genetic locus that encodes the uptake capability in Salmonella is fra. This fra locus has five genes: fraR, fraB a fructose-asparagine deglycase, fraD a sugar kinase, fraA a fructose-asparagine transporter, and fraE a L-asparaginase. Notably, mutations in fraB cause the buildup of the toxic intermediate 6-phosphofuctose-aspartate (6-P-F-Asp). The buildup of 6-P-F-Asp has a bacteriostatic effect on fraB mutant cells, making FraB a potential drug target.

References

  1. ↑ Hutson RG, Kitoh T, Moraga Amador DA, Cosic S, Schuster SM, Kilberg MS (May 1997). "Amino acid control of asparagine synthetase: relation to asparaginase resistance in human leukemia cells". The American Journal of Physiology. 272 (5 Pt 1): C1691-9. doi:10.1152/ajpcell.1997.272.5.C1691. PMID   9176161.
  2. 1 2 3 4 5 6 7 8 9 Balasubramanian MN, Butterworth EA, Kilberg MS (April 2013). "Asparagine synthetase: regulation by cell stress and involvement in tumor biology". American Journal of Physiology. Endocrinology and Metabolism. 304 (8): E789-99. doi:10.1152/ajpendo.00015.2013. PMC   3625782 . PMID   23403946.
  3. 1 2 3 4 5 6 Prager MD, Bachynsky N (April 1968). "Asparagine synthetase in asparaginase resistant and susceptible mouse lymphomas". Biochemical and Biophysical Research Communications. 31 (1): 43–7. doi:10.1016/0006-291x(68)90028-4. PMID   4869945.
  4. 1 2 3 4 Larsen TM, Boehlein SK, Schuster SM, Richards NG, Thoden JB, Holden HM, Rayment I (December 1999). "Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product". Biochemistry. 38 (49): 16146–57. doi:10.1021/bi9915768. PMID   10587437.
  5. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Richards NG, Kilberg MS (July 2006). "Asparagine synthetase chemotherapy". Annual Review of Biochemistry. 75: 629–54. doi:10.1146/annurev.biochem.75.103004.142520. PMC   3587692 . PMID   16756505.
  6. 1 2 3 4 5 6 Gaufichon L, Reisdorf-Cren M, Rothstein SJ, Chardon F, Suzuki A (September 2010). "Biological functions of asparagine synthetase in plants". Plant Science. 179 (3): 141–153. doi:10.1016/j.plantsci.2010.04.010.
  7. 1 2 3 4 Richards NG, Schuster SM (November 1998). "Mechanistic issues in asparagine synthetase catalysis". Advances in Enzymology and Related Areas of Molecular Biology. Vol. 72. pp. 145–98. doi:10.1002/9780470123188.ch5. ISBN   9780470123188. PMID   9559053.
  8. ↑ Heng HH, Shi XM, Scherer SW, Andrulis IL, Tsui LC (1994). "Refined localization of the asparagine synthetase gene (ASNS) to chromosome 7, region q21.3, and characterization of the somatic cell hybrid line 4AF/106/KO15". Cytogenetics and Cell Genetics. 66 (2): 135–8. doi:10.1159/000133685. hdl: 10722/42532 . PMID   7904551.
  9. ↑ Chan WK, Lorenzi PL, Anishkin A, Purwaha P, Rogers DM, Sukharev S, Rempe SB, Weinstein JN (June 2014). "The glutaminase activity of L-asparaginase is not required for anticancer activity against ASNS-negative cells". Blood. 123 (23): 3596–606. doi:10.1182/blood-2013-10-535112. PMC   4047499 . PMID   24659632.
  10. 1 2 Prager MD, Bachynsky N (September 1968). "Asparagine synthetase in normal and malignant tissues: correlation with tumor sensitivity to asparaginase". Archives of Biochemistry and Biophysics. 127 (1): 645–54. doi:10.1016/0003-9861(68)90273-7. PMID   4880551.
  11. ↑ Horowitz B, Madras BK, Meister A, Old LJ, Boyes EA, Stockert E (May 1968). "Asparagine synthetase activity of mouse leukemias". Science. 160 (3827): 533–5. Bibcode:1968Sci...160..533H. doi:10.1126/science.160.3827.533. PMID   5689413. S2CID   39734239.
  12. ↑ Haskell CM, Canellos GP (October 1969). "l-asparaginase resistance in human leukemia--asparagine synthetase". Biochemical Pharmacology. 18 (10): 2578–80. doi:10.1016/0006-2952(69)90375-x. PMID   4935103.
  13. ↑ Aslanian AM, Fletcher BS, Kilberg MS (July 2001). "Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells". The Biochemical Journal. 357 (Pt 1): 321–8. doi:10.1042/bj3570321. PMC   1221958 . PMID   11415466.
  14. ↑ Khater F, Lajoie M, Langlois S, Healy J, Cellot S, Richer C, Beaulieu P, St-Onge P, Sailloir V, Minden M, Marzouki M, Krajinovic M, Bittencourt H, Sinnett D (2017). "KMT2E-ASNS: a novel relapse-specific fusion gene in early T-cell precursor acute lymphoblastic leukemia". Blood. 129 (12): 1729–1732. doi:10.1182/blood-2016-10-744219. PMC   5374844 . PMID   28069604.
  15. ↑ Andrulis IL, Chen J, Ray PN (July 1987). "Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells". Molecular and Cellular Biology. 7 (7): 2435–43. doi:10.1128/MCB.7.7.2435. PMC   365375 . PMID   2886907.
  16. 1 2 3 Lorenzi PL, Weinstein JN (January 2009). "Asparagine synthetase: a new potential biomarker in ovarian cancer". Drug News & Perspectives. 22 (1): 61–4. doi:10.1358/dnp.2009.22.1.1303820. PMC   4096155 . PMID   19209300.
  17. 1 2 Patrikainen L, Porvari K, Kurkela R, Hirvikoski P, Soini Y, Vihko P (February 2007). "Expression profiling of PC-3 cell line variants and comparison of MIC-1 transcript levels in benign and malignant prostate". European Journal of Clinical Investigation. 37 (2): 126–33. doi:10.1111/j.1365-2362.2007.01763.x. PMID   17217378. S2CID   29946047.
  18. 1 2 3 4 5 Ameri K, Luong R, Zhang H, Powell AA, Montgomery KD, Espinosa I, Bouley DM, Harris AL, Jeffrey SS (February 2010). "Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype". British Journal of Cancer. 102 (3): 561–9. doi:10.1038/sj.bjc.6605491. PMC   2805847 . PMID   20051957.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.