Purinosome

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The synthesis of IMP. The color scheme is as follows: enzymes, coenzymes, substrate names, metal ions, inorganic molecules Nucleotides syn1.png
The synthesis of IMP.
The color scheme is as follows: enzymes, coenzymes, substrate names, metal ions, inorganic molecules

The purinosome is a putative multi-enzyme complex that carries out de novo purine biosynthesis within the cell. It is postulated to include all six of the human enzymes identified as direct participants in this ten-step biosynthetic pathway converting phosphoribosyl pyrophosphate to inosine monophosphate:

Contents

Step(s)SymbolDescription
1PPAT phosphoribosylpyrophosphate amidotransferase
2,3,5GARTtrifunctional phosphoribosylglycinamide formyltransferase/phosphoribosylglycinamide synthetase/phosphoribosylaminoimidazole synthetase
4PFAS phosphoribosylformylglycinamidine synthase
6,7PAICSbifunctional phosphoribosylaminoimidazole carboxylase
8ADSL adenylosuccinate lyase
9,10ATICbifunctional 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase

History

Hypothesis

The enzymes of the multi-step de novo purine biosynthesis pathway have been postulated to form a multi-enzyme complex to facilitate substrate channeling between each enzyme of the pathway. Slight variations of the pathway exists between phyla; however, there are 13 enzymes that can be considered part of this biosynthetic pathway. [1] Several individual enzymatic functions have consolidated onto single bifunctional or trifunctional polypeptide chains in higher organisms, suggesting stable physical interactions exist between enzymes. [2] [3] The functional consolidation of steps 2,3, and 5 of the pathway into a single enzyme in higher organisms such as humans suggests physical local proximity of the enzyme for step 4 to the trifunctional enzyme. [2] [4] [5]

Evidence for a complex

The purine biosynthesis enzymes can be co-purified under certain conditions. [6] [7] A complex of two particular pathway enzymes GART and ATIC can be isolated with cofactor production enzyme C1THF synthase and SHMT1. [8] Kinetic studies show evidence of substrate channeling between PPAT and GART, but evidence could not be obtained for their physical protein-protein interaction. [9] Thus far, isolation of a multienzyme complex inclusive of all purine biosynthesis enzymes has not been achieved. In yeast, some enzymes implicated in de novo purine biosynthesis pathway were shown to be able to form mesoscale punctuations in cells. [10] In E. coli, binary protein-protein interaction screening have shown the potential existence of a bacterial purinosome equivalent with a different architecture than in mammalian cells [11] [12]

Purinosome macrobodies

Macrobodies composed of purinosome members. Purine biosynthesis enzymes cluster into discrete intracellular bodies when transiently expressed as fusions to enhanced green fluorescent protein (EGFP) in HeLa cells. FGAMS is an alternate name for PFAS. Fgams ppat egfp puncta.png
Macrobodies composed of purinosome members.
Purine biosynthesis enzymes cluster into discrete intracellular bodies when transiently expressed as fusions to enhanced green fluorescent protein (EGFP) in HeLa cells. FGAMS is an alternate name for PFAS.

Purinosome macrobodies (also may be referred to as bodies, clusters, foci, puncta) describe the assembly of fluorescent-tagged human purine biosynthetic enzymes into bodies visible by fluorescence microscopy. The purinosome body theory states that purinosome bodies are assembled from proteins normally dispersed in the cell, and this assembly manifests when the demand for purines exceeds the amount supplied by the purine salvage pathway, such as when the extracellular medium is depleted of purines. In addition to the 6 purine biosynthesis pathway proteins, purinosome macrobodies are composed of at least 10 additional proteins not involved in purine biosynthesis. Due to the nature of their expression and association with cellular stress response proteins, purinosome macrobodies may actually be aggregated protein bodies.

Initial discovery

The human purinosome was thought to have been identified in 2008 by the observation that transiently expressed GFP fusion constructs of purine biosynthesis proteins form macrobodies. [14] [15] A folate enzyme not directly involved in the purine biosynthesis pathway, 5,10-methenyltetrahydrofolate synthase (MTHFS), was later found to be part of purinosome macrobodies by the same approach. [16] The biological relevance of this folate enzyme's inclusion to the purinosome macrobody is unclear: while it provides substrate for a trifunctional folate enzyme C1THF synthase to generate a key cofactor for purine biosynthesis, C1THF synthase is not a part of purinosome macrobodies. [14] Curiously, hypoxanthine levels do not alter purinosome macrobodies, [14] but adenosine or guanosine addition suppresses formation of macromolecular bodies formed by the folate enzyme. [16]

Aggregation

Later studies in 2013 support the interpretation that those macrobodies could be artifacts of aggregated proteins that commonly result from fusion protein expression. [13] Characteristics of purinosome bodies were found to be shared between those of canonical protein aggregates, such as induction by peroxide. While purinosome bodies were also found to be associated with early cell death, it is unclear whether the bodies were a cause of that stress or rather an indicator of stressed cells.

Discrepancies

Inhibition of microtubule polymerization with nocodazole blocks formation of the purinosome macrobodies, and reduces the flux of de novo purine biosynthesis. [17] However, nocodazole also blocks formation of aggresomes, complicating interpretation of these observations. Partial inhibition of casein kinase 2 by small molecule inhibitors - 4,5,6,7-tetrabromo-1H-benzimidazole (TBI), 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), tetrabromocinammic acid (TBCA) or ellagic acid - was found to induce purinosome macrobody formation, while another inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB) induced purinosome macrobody formation at low concentration but not at high concentration, and caused the dissociation of the bodies formed in response to DMAT. [18] Complicating the interpretation of these data, inhibition of casein kinase 2 is also known to disrupt hundreds of cellular processes, among them being protein homeostasis which regulates protein aggregation.

Additional members of purinosome macrobodies

Proteins excluded from purinosome macrobodies

Related Research Articles

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Nucleotides are organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules within all life-forms on Earth. Nucleotides are obtained in the diet and are also synthesized from common nutrients by the liver.

<span class="mw-page-title-main">Dihydrofolate reductase</span> Mammalian protein found in humans

Dihydrofolate reductase, or DHFR, is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as an electron donor, which can be converted to the kinds of tetrahydrofolate cofactors used in 1-carbon transfer chemistry. In humans, the DHFR enzyme is encoded by the DHFR gene. It is found in the q14.1 region of chromosome 5.

A nucleoside triphosphate is a nucleoside containing a nitrogenous base bound to a 5-carbon sugar, with three phosphate groups bound to the sugar. They are the molecular precursors of both DNA and RNA, which are chains of nucleotides made through the processes of DNA replication and transcription. Nucleoside triphosphates also serve as a source of energy for cellular reactions and are involved in signalling pathways.

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">Pterin</span> Chemical compound

Pterin is a heterocyclic compound composed of a pteridine ring system, with a "keto group" and an amino group on positions 4 and 2 respectively. It is structurally related to the parent bicyclic heterocycle called pteridine. Pterins, as a group, are compounds related to pterin with additional substituents. Pterin itself is of no biological significance.

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

Formylation refers to any chemical processes in which a compound is functionalized with a formyl group (-CH=O). In organic chemistry, the term is most commonly used with regards to aromatic compounds. In biochemistry the reaction is catalysed by enzymes such as formyltransferases.

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

Phosphoribosyl pyrophosphate (PRPP) is a pentose phosphate. It is a biochemical intermediate in the formation of purine nucleotides via inosine-5-monophosphate, as well as in pyrimidine nucleotide formation. Hence it is a building block for DNA and RNA. The vitamins thiamine and cobalamin, and the amino acid tryptophan also contain fragments derived from PRPP. It is formed from ribose 5-phosphate (R5P) by the enzyme ribose-phosphate diphosphokinase:

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

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

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

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

Phosphoribosylformylglycinamidine cyclo-ligase is the fifth enzyme in the de novo synthesis of purine nucleotides. It catalyzes the reaction to form 5-aminoimidazole ribotide (AIR) from formylglycinamidine-ribonucleotide FGAM. This reaction closes the ring and produces a 5-membered imidazole ring of the purine nucleus (AIR):

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<span class="mw-page-title-main">Ribose-phosphate diphosphokinase</span> Class of enzymes

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<span class="mw-page-title-main">Trifunctional purine biosynthetic protein adenosine-3</span> Mammalian protein found in Homo sapiens

Trifunctional purine biosynthetic protein adenosine-3 is an enzyme that in humans is encoded by the GART gene.

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Bifunctional purine biosynthesis protein PURH is a protein that in humans is encoded by the ATIC gene.

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<span class="mw-page-title-main">MTHFD1</span>

Methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1) is a gene located in humans on chromosome 14 that encodes a protein, C-1-tetrahydrofolate synthase, cytoplasmic also known as C1-THF synthase, with three distinct enzymatic activities.

<span class="mw-page-title-main">5-Aminoimidazole ribotide</span> Chemical compound

5′-Phosphoribosyl-5-aminoimidazole is a biochemical intermediate in the formation of purine nucleotides via inosine-5-monophosphate, and hence is a building block for DNA and RNA. The vitamins thiamine and cobalamin also contain fragments derived from AIR. It is an intermediate in the adenine pathway and is synthesized from 5′-phosphoribosylformylglycinamidine by AIR synthetase.

<span class="mw-page-title-main">Phosphoribosylglycinamide formyltransferase</span>

Phosphoribosylglycinamide formyltransferase (EC 2.1.2.2), also known as glycinamide ribonucleotide transformylase (GAR Tfase), is an enzyme with systematic name 10-formyltetrahydrofolate:5'-phosphoribosylglycinamide N-formyltransferase. This enzyme catalyses the following chemical reaction

<span class="mw-page-title-main">Stephen J. Benkovic</span> American chemist

Stephen James Benkovic is an American chemist known for his contributions to the field of enzymology. He holds the Evan Pugh University Professorship and Eberly Chair in Chemistry at The Pennsylvania State University. He has developed boron compounds that are active pharmacophores against a variety of diseases. Benkovic has concentrated on the assembly and kinetic attributes of the enzymatic machinery that performs DNA replication, DNA repair, and purine biosynthesis.

References

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  8. ↑ Smith, G. K.; Mueller, W. T.; Wasserman, G. F.; Taylor, W. D.; Benkovic, S. J. (1980). "Characterization of the enzyme complex involving the folate-requiring enzymes of de novo purine biosynthesis". Biochemistry. 19 (18): 4313–21. doi:10.1021/bi00559a026. PMID   7417406.
  9. ↑ J. Rudolph; J. Stubbe (1995). "Investigation of the Mechanism of Phosphoribosylamine Transfer from Glutamine Phosphoribosylpyrophosphate Amidotransferase to Glycinamide Ribonucleotide Synthetase". Biochemistry. 34 (7): 2241–2250. doi:10.1021/bi00007a019. PMID   7532005.
  10. ↑ Noree, Chalongrat; Begovich, Kyle; Samilo, Dane; Broyer, Risa; Monfort, Elena; Wilhelm, James E. (2019-10-01). "A quantitative screen for metabolic enzyme structures reveals patterns of assembly across the yeast metabolic network". Molecular Biology of the Cell. 30 (21): 2721–2736. doi:10.1091/mbc.E19-04-0224. ISSN   1939-4586. PMC   6761767 . PMID   31483745.
  11. ↑ Gedeon, Antoine; Karimova, Gouzel; Ayoub, Nour; Dairou, Julien; Giai Gianetto, Quentin; Vichier-Guerre, Sophie; Vidalain, Pierre-Olivier; Ladant, Daniel; Munier-Lehmann, Hélène (June 2023). "Interaction network among de novo purine nucleotide biosynthesis enzymes in Escherichia coli". The FEBS Journal. 290 (12): 3165–3184. doi:10.1111/febs.16746. ISSN   1742-464X. PMID   36748301.
  12. ↑ Ayoub, Nour; Gedeon, Antoine; Munier-Lehmann, Hélène (2024-02-20). "A journey into the regulatory secrets of the de novo purine nucleotide biosynthesis". Frontiers in Pharmacology. 15. doi: 10.3389/fphar.2024.1329011 . ISSN   1663-9812. PMC   10912719 . PMID   38444943.
  13. 1 2 3 4 5 Alice Zhao; Mark Tsechansky; Jagannath Swaminathan; Lindsey Cook; Andrew D. Ellington; Edward M. Marcotte (2013-02-06). "Transiently Transfected Purine Biosynthetic Enzymes Form Stress Bodies". PLOS ONE. 8 (2): e56203. Bibcode:2013PLoSO...856203Z. doi: 10.1371/journal.pone.0056203 . PMC   3566086 . PMID   23405267.
  14. 1 2 3 4 5 Songon An; et al. (2008). "Reversible Compartmentalization of de Novo Purine Biosynthetic Complexes in Living Cells". Science. 320 (5872): 103–106. Bibcode:2008Sci...320..103A. doi:10.1126/science.1152241. PMID   18388293. S2CID   24119538.
  15. ↑ Hong Zhao; Jarrod B. French; Ye Fang; Stephen J. Benkovic (2013-04-03). "The purinosome, a multi-protein complex involved in the de novo biosynthesis of purines in humans". Chem. Commun. 285 (15): 11093–11099. doi:10.1039/c3cc41437j. PMC   3877848 . PMID   23575936.
  16. 1 2 3 Martha S. Field; Donald D. Anderson; Patrick J. Stover (2011). "Mthfs is an essential gene in mice and a component of the purinosome". Front Genet. 2 (36): 36. doi: 10.3389/fgene.2011.00036 . PMC   3268590 . PMID   22303332.
  17. ↑ Songon An; Yijun Deng; John W. Tomsho; Minjoung Kyoung; Stephen J. Benkovic (2010-07-20). "Microtubule-assisted mechanism for functional metabolic macromolecular complex formation". Proc. Natl. Acad. Sci. U.S.A. 107 (29): 12872–12876. Bibcode:2010PNAS..10712872A. doi: 10.1073/pnas.1008451107 . PMC   2919939 . PMID   20615962.
  18. ↑ Songon An; Minjoung Kyoung; Jasmina J. Allen; Kevan M. Shokat; Stephen J. Benkovic (2010-04-09). "Dynamic regulation of a metabolic multi-enzyme complex by protein kinase CK2". J Biol Chem. 285 (15): 11093–11099. doi: 10.1074/jbc.M110.101139 . PMC   2856985 . PMID   20157113.
  19. 1 2 3 4 5 6 7 8 9 10 11 12 13 French, J. B.; et al. (2013-01-28). "Hsp70/Hsp90 chaperone machinery is involved in the assembly of the purinosome". Proceedings of the National Academy of Sciences. 110 (7): 2528–2533. Bibcode:2013PNAS..110.2528F. doi: 10.1073/pnas.1300173110 . ISSN   0027-8424. PMC   3574928 . PMID   23359685.
  20. ↑ Yamada, Seiya; Sato, Ayaka; Sakakibara, Shin-Ichi (2020-05-22). "Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex". iScience. 23 (5): 101058. Bibcode:2020iSci...23j1058Y. doi:10.1016/j.isci.2020.101058. ISSN   2589-0042. PMC   7186558 . PMID   32344379.
  21. ↑ Alice Zhao; Mark Tsechansky; Jagannath Swaminathan; Lindsey Cook; Andrew Ellington; Edward Marcotte. "By the same standards, prior work may not either" (PDF). Archived from the original (PDF) on 4 February 2014. Retrieved 20 April 2013.
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