isocitrate lyase

This enzyme belongs to the family of lyases, specifically the oxo-acid-lyases, which cleave carbon-carbon bonds. Other enzymes also belong to this family including carboxyvinyl-carboxyphosphonate phosphorylmutase ({{EC number|2.7.8.23}}) which catalyses the conversion of 1-carboxyvinyl carboxyphosphonate to 3-(hydrohydroxyphosphoryl) pyruvate carbon dioxide, and phosphoenolpyruvate mutase ({{EC number|5.4.2.9}}), which is involved in the biosynthesis of phosphinothricin tripeptide antibiotics.

During catalysis, isocitrate is deprotonated, and an aldol cleavage results in the release of succinate and glyoxylate. This reaction mechanism functions much like that of aldolase in glycolysis, where a carbon-carbon bond is cleaved and an aldehyde is released.{{cite book|vauthors=Garrett R, Grisham CN|title=Biochemistry|url=https://archive.org/details/biochemistry00rhga|url-access=limited|year=2008|publisher=Brooks Cole|isbn=978-0-495-10935-8|pages=[https://archive.org/details/biochemistry00rhga/page/n625 588]}}

File:ICL catalyzed rxn.png

In the glyoxylate cycle, malate synthase then catalyzes the condensation of glyoxylate and acetyl-CoA to form malate so the cycle can continue.

ICL competes with isocitrate dehydrogenase, an enzyme found in the TCA cycle, for isocitrate processing. Flux through these enzymes is controlled by phosphorylation of isocitrate dehydrogenase, which has a much higher affinity for isocitrate as compared to ICL.{{cite journal | vauthors = Cozzone AJ | title = Regulation of acetate metabolism by protein phosphorylation in enteric bacteria | journal = Annual Review of Microbiology | volume = 52 | pages = 127–64 | year = 1998 | pmid = 9891796 | doi = 10.1146/annurev.micro.52.1.127 }} Deactivation of isocitrate dehydrogenase by phosphorylation thus leads to increased isocitrate channeling through ICL, as seen when bacteria are grown on acetate, a two-carbon compound.

As of 2023, multiple structures of ICL have been solved. These include one structure from Pseudomonas aeruginosa (PDB accession code {{PDB link|6G1O}}), one structure from Fusarium graminearum ({{PDB link|5E9H}}), one structure from fungus Aspergillus nidulans ({{PDB link|1DQU}}), one structure from Yersinia pestis ({{PDB link|3LG3}}), one structure from Burkholderia pseudomallei ({{PDB link|3I4E}}), one structure from Escherichia coli ({{PDB link|1IGW}}), two structures from Magnaporthe oryzae ({{PDB link|5E9F}} and {{PDB link|5E9G}}), four structures from Brucella melitensis ({{PDB link|3P0X}}, {{PDB link|3OQ8}}, {{PDB link|3EOL}} and {{PDB link|3E5B}}) and eleven structures from Mycobacterium tuberculosis ({{PDB link|1F61}}, {{PDB link|1F8I}}, {{PDB link|1F8M}}, {{PDB link|6C4A}}, {{PDB link|6C4C}}, {{PDB link|5DQL}}, {{PDB link|6EDW}}, {{PDB link|6EDZ}}, {{PDB link|6EE1}}, {{PDB link|6XPP}} and {{PDB link|8G8K}}).

ICL is composed of four identical chains and requires a Mg²⁺ or Mn²⁺ and a thiol for activity. In Escherichia coli, Lys-193, Lys-194, Cys-195, His-197, and His-356 are thought to be catalytic residues, while His-184 is thought to be involved in the assembly of the tetrameric enzyme.{{cite journal | vauthors = Rehman A, McFadden BA | title = Lysine 194 is functional in isocitrate lyase from Escherichia coli | journal = Current Microbiology | volume = 35 | issue = 1 | pages = 14–7 | date = July 1997 | pmid = 9175553 | doi = 10.1007/s002849900203 | s2cid = 23972776 }}

Between prokaryotes and eukaryotes, a difference in ICL structure is the addition of approximately 100 amino acids near the center of the eukaryotic enzyme. In eukaryotes, the additional amino acids are thought to function in the localization of ICL to single-membrane-bound organelles called glyoxysomes.{{cite journal | vauthors = Eastmond PJ, Graham IA | title = Re-examining the role of the glyoxylate cycle in oilseeds | journal = Trends in Plant Science | volume = 6 | issue = 2 | pages = 72–8 | date = February 2001 | pmid = 11173291 | doi = 10.1016/S1360-1385(00)01835-5 | bibcode = 2001TPS.....6...72E }} These additional amino acids account for the difference in molecular mass: the prokaryotic ICL is 48kDa, while the eukaryotic ICL is 67 kDa. Only one cysteine residue is conserved between the sequences of the fungal, plant and bacterial enzymes; it is located in the middle of a conserved hexapeptide.

Most ICLs that have been characterised to date contain only one domain (the catalytic domain). However, in the isoform 2 of M. tuberculosis ICL, two domains were found.{{cite journal|author1=Bhusal, R. P. |author2=Jiao, W. |author3=Kwai, B. X. C. |author4=Reynisson, J. |author5=Collins, A. J. |author6=Sperry, J. |author7=Bashiri, G. |author8=Leung, I. K. H. |title=Acetyl-CoA-Mediated Activation of Mycobacterium tuberculosis Isocitrate Lyase 2 |journal=Nature Communications|date=Oct 2019|volume=10|issue=1|page=4639|pmid=31604954|doi=10.1038/s41467-019-12614-7|pmc=6788997|bibcode=2019NatCo..10.4639B }} Through structural and kinetic studies, the C-terminal domain was found to be a regulatory domain, which dimerises with the corresponding C-terminal domain from another subunit (of the ICL2 tetramer) upon the binding of acetyl coenzyme A to activate the catalytic activity of the enzyme.

In M. tuberculosis H37Rv (a commonly used laboratory strain), the gene that encodes ICL2 was split into two open reading frames (rv1915 and rv1916), thus encoding Rv1915 (ICL2a) and Rv1916 (ICL2b) respectively. The biological functions of Rv1915 (ICL2a) and Rv1916 (ICL2b) are poorly understood. Rv1915 and rv1916 were initially characterized as pseudogenes.{{cite journal | vauthors = Höner Zu Bentrup K, Miczak A, Swenson DL, Russell DG | title = Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis | journal = Journal of Bacteriology | volume = 181 | issue = 23 | pages = 7161–7 | date = December 1999 | pmid = 10572116 | pmc = 103675 | doi = 10.1128/JB.181.23.7161-7167.1999}} An in silico study in 2019 predicted that Rv1916 (ICL2b) could be involved in the synthesis of secondary metabolites.{{cite journal | vauthors = Antil M, Sharma J, Brissonnet Y, Choudhary M, Gouin S, Gupta V | title = Structure function insights into elusive Mycobacterium tuberculosis protein Rv1916 | journal = International Journal of Biological Macromolecules | volume = 141| date = September 2019 | pmid = 31505209 | doi = 10.1016/j.ijbiomac.2019.09.038 | pages = 927–936 | s2cid = 202556684 | url = https://hal.archives-ouvertes.fr/hal-03003095/file/Preprint.pdf }} In vitro studies showed that both Rv1915 (ICL2a) and Rv1916 (ICL2b) may be able to catalyze the conversion of isocitrate to form succinate and glyoxylate.{{cite journal | vauthors = Antil M, Gupta V | title = Rv1915 and Rv1916 from Mycobacterium tuberculosis H37Rv form in vitro protein-protein complex | journal = Biochimica et Biophysica Acta (BBA) - General Subjects | volume = 1866 | date = June 2022 | issue = 6 | pmid = 35307510 | doi = 10.1016/j.bbagen.2022.130130 | page = 130130 }} However, a recent structural and biochemical study showed that Rv1916 (ICL2b) does not have ICL activity.{{cite journal |vauthors = Huang EY, Kwai BX, Bhusal RP, Bashiri G, Leung, IK | title = Mycobacterium tuberculosis Rv1916 is an Acetyl-CoA-Binding Protein | journal = ChemBioChem | volume = 24| date = May 2023 | issue = 14 | pmid = 37211532 | doi = 10.1002/cbic.202300162 | pages = e202300162 | doi-access = free | hdl = 2292/64343 | hdl-access = free }} Instead, the study showed that Rv1916 (ICL2b) is a acetyl-CoA-binding protein with unknown biological function.

Several assays were developed to study the enzyme kinetics and inhibition of ICL. The most frequently used assays involved the use of chemical or enzyme-coupled ultraviolet–visible (UV/vis) spectroscopy to measure the amount of glyoxylate that is being formed. For example, glyoxylate can be reacted with phenylhydrazine to form hydrazone that can be analysed by UV/vis spectroscopy.{{cite journal | title = Proceedings of the Biochemical Society | journal = The Biochemical Journal | volume = 72 | issue = 1 | pages = 1P–13P | date = May 1959 | pmid = 16748793 | pmc = 1196904 | doi = 10.1042/bj0720001P }} Alternatively, lactate dehydrogenase can be used to catalyse the reduction of glyoxylate to glycolate in the presence of nicotinamide adenine dinucleotide (NADH), which is a cosubstrate of lactate dehydrogenase. During the reaction, NADH is being oxidised to NAD⁺. The decrease in NADH concentration can then measured by UV/vis spectroscopy using a dye. In additional to spectroscopic techniques, biophysical techniques including native non-denaturing mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy have also been applied to study ICL.{{cite journal | vauthors = Pham TV, Murkin AS, Moynihan MM, Harris L, Tyler PC, Shetty N, Sacchettini JC, Huang HL, Meek TD | display-authors = 6 | title = Mycobacterium tuberculosis | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 114 | issue = 29 | pages = 7617–7622 | date = July 2017 | pmid = 28679637 | pmc = 5530696 | doi = 10.1073/pnas.1706134114 | doi-access = free }}{{cite journal | vauthors = Bhusal RP, Patel K, Kwai BX, Swartjes A, Bashiri G, Reynisson J, Sperry J, Leung IK | display-authors = 6 | title = Mycobacterium tuberculosis isocitrate lyase inhibitors | journal = MedChemComm | volume = 8 | issue = 11 | pages = 2155–2163 | date = November 2017 | pmid = 30108733 | pmc = 6072051 | doi = 10.1039/C7MD00456G }}

The ICL enzyme has been found to be functional in various archaea, bacteria, protists, plants, fungi, and nematodes.{{cite journal | vauthors = Kondrashov FA, Koonin EV, Morgunov IG, Finogenova TV, Kondrashova MN | title = Evolution of glyoxylate cycle enzymes in Metazoa: evidence of multiple horizontal transfer events and pseudogene formation | journal = Biology Direct | volume = 1 | issue = 31 | page = 31 | date = October 2006 | pmid = 17059607 | pmc = 1630690 | doi = 10.1186/1745-6150-1-31 | doi-access = free }} Although the gene has been found in genomes of nematodes and cnidaria, it has not been found in the genomes of placental mammals.

By diverting isocitrate from the TCA cycle, the actions of ICL and malate synthase in the glyoxylate cycle result in the net assimilation of carbon from 2-carbon compounds.{{cite journal | vauthors = Kornberg HL, Krebs HA | title = Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle | journal = Nature | volume = 179 | issue = 4568 | pages = 988–91 | date = May 1957 | pmid = 13430766 | doi = 10.1038/179988a0 | bibcode = 1957Natur.179..988K | s2cid = 40858130 }} Thus, while the TCA cycle yields no net carbon assimilation, the glyoxylate cycle generates intermediates that can be used to synthesize glucose (via gluconeogenesis), plus other biosynthetic products. As a result, organisms that use ICL and malate synthase are able to synthesize glucose and its metabolic intermediates from acetyl-CoA derived from acetate or from the degradation of ethanol, fatty acids, or poly-β-hydroxybutyrate. This function is especially important for higher plants when using seed oils. In germinating seeds, the breakdown of oils generates acetyl-CoA. This serves as a substrate for the glyoxylate cycle, which generates intermediates which serve as a primary nutrient source prior to the beginning of production of sugars by photosynthesis.

In M. tuberculosis, ICL isoforms 1 and 2 also play the role of methylisocitrate lyase, converting methylisocitrate into succinate and pyruvate.{{cite journal | vauthors = Gould TA, van de Langemheen H, Muñoz-Elías EJ, McKinney JD, Sacchettini JC | title = Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis | journal = Molecular Microbiology | volume = 61 | issue = 4 | pages = 940–7 | date = August 2006 | pmid = 16879647 | doi = 10.1111/j.1365-2958.2006.05297.x | s2cid = 26099043 | doi-access = free }} This is important because the methylcitrate cycle is key for the survival of the bacteria on odd-chain fatty acids.{{cite journal | vauthors = Eoh H, Rhee KY | title = Methylcitrate cycle defines the bactericidal essentiality of isocitrate lyase for survival of Mycobacterium tuberculosis on fatty acids | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 13 | pages = 4976–81 | date = April 2014 | pmid = 24639517 | pmc = 3977286 | doi = 10.1073/pnas.1400390111 | bibcode = 2014PNAS..111.4976E | doi-access = free }}

ICL has found to be important in human, animal, and plant pathogenesis. For several agricultural crops including cereals, cucumbers, and melons, increased expression of the gene encoding ICL is important for fungal virulence. For instance, increased gene expression of icl1 has been seen in the fungus Leptosphaeria maculans upon infection of canola. Inactivation of the icl1 gene leads to reduced pathogenicity of the fungus, which is thought to be a result of the inability of the fungus to use carbon sources provided by the plant.{{cite journal | vauthors = Idnurm A, Howlett BJ | title = Isocitrate lyase is essential for pathogenicity of the fungus Leptosphaeria maculans to canola (Brassica napus) | journal = Eukaryotic Cell | volume = 1 | issue = 5 | pages = 719–24 | date = October 2002 | pmid = 12455691 | pmc = 126752 | doi = 10.1128/EC.1.5.719-724.2002 }}

Additionally, upregulation of the glyoxylate cycle has been seen for pathogens that attack humans. This is the case for fungi such as Candida albicans, which inhabits the skin, mouth, GI tract, gut and vagina of mammals and can lead to systemic infections of immunocompromised patients; as well as for the bacterium Mycobacterium tuberculosis, the major causative agent of tuberculosis.{{cite journal | vauthors = Lorenz MC, Bender JA, Fink GR | title = Transcriptional response of Candida albicans upon internalization by macrophages | journal = Eukaryotic Cell | volume = 3 | issue = 5 | pages = 1076–87 | date = October 2004 | pmid = 15470236 | pmc = 522606 | doi = 10.1128/EC.3.5.1076-1087.2004 }}{{cite journal | vauthors = Srivastava V, Jain A, Srivastava BS, Srivastava R | title = Selection of genes of Mycobacterium tuberculosis upregulated during residence in lungs of infected mice | journal = Tuberculosis | volume = 88 | issue = 3 | pages = 171–7 | date = May 2008 | pmid = 18054522 | doi = 10.1016/j.tube.2007.10.002 }} In this latter case, ICL has been found to be essential for survival in the host.{{cite journal | vauthors = Muñoz-Elías EJ, McKinney JD | title = Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence | journal = Nature Medicine | volume = 11 | issue = 6 | pages = 638–44 | date = June 2005 | pmid = 15895072 | pmc = 1464426 | doi = 10.1038/nm1252 }} Thus, ICL is a current inhibition target for therapeutic treatments of tuberculosis.{{cite journal | vauthors = Bhusal RP, Bashiri G, Kwai BX, Sperry J, Leung IK | title = Targeting isocitrate lyase for the treatment of latent tuberculosis | journal = Drug Discovery Today | volume = 22 | issue = 7 | pages = 1008–1016 | date = July 2017 | pmid = 28458043 | doi = 10.1016/j.drudis.2017.04.012 }}

Because of its use by pathogenic fungi and bacteria, specific inhibitors are being sought for ICL and malate synthase. Although some inhibitors have already been identified, including itaconate, itaconic anhydride, bromopyruvate, nitropropionate, oxalate, and malate, these are non-specific and would also inhibit other enzymes essential for host function.{{cite journal | vauthors = Krátký M, Vinšová J | title = Advances in mycobacterial isocitrate lyase targeting and inhibitors | journal = Current Medicinal Chemistry | volume = 19 | issue = 36 | pages = 6126–37 | date = Dec 2012 | pmid = 23092127 | doi = 10.2174/0929867311209066126 }}{{cite journal | vauthors = Lee YV, Wahab HA, Choong YS | title = Potential inhibitors for isocitrate lyase of Mycobacterium tuberculosis and non-M. tuberculosis: a summary | journal = BioMed Research International | volume = 2015 | page = 895453 | date = 2015 | pmid = 25649791 | pmc = 4306415 | doi = 10.1155/2015/895453 | doi-access = free }} More research is needed to identify inhibitors that selectively target enzymes in the glyoxylate cycle.

• Glyoxylate cycle
• Isocitrate lyase family

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{{refbegin}}

• {{cite journal | vauthors = McFadden BA, Howes WV | year = 1963 | title = Crystallisation and some properties of isocitrate lyase from Pseudomonas indigofera | journal = J. Biol. Chem. | volume = 238 | issue = 5 | pages = 1737–1742 | doi = 10.1016/S0021-9258(18)81129-2 | doi-access = free }}
• {{cite journal | vauthors = Shiio I, Shiio T, Mcfadden BA | title = Isocitrate lyase from Pseudomonas indigofera I. Preparation, amino acid composition and molecular weight | journal = Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis | volume = 96 | pages = 114–22 | date = January 1965 | pmid = 14285253 | doi = 10.1016/0005-2787(65)90615-5 }}
• {{cite journal | vauthors = Vickery HB | title = A suggested new nomenclature for the isomers of isocitric acid | journal = The Journal of Biological Chemistry | volume = 237 | pages = 1739–41 | date = June 1962 | issue = 6 | doi = 10.1016/S0021-9258(19)73928-3 | pmid = 13925783 | doi-access = free }}

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{{Carbon-carbon lyases}}

{{Enzymes}}

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{{DEFAULTSORT:Isocitrate Lyase}}

Category:EC 4.1.3

Category:Enzymes of known structure